Biochemistry

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 Chapter 1
Introduction to Biochemistry

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As the name indicates, biochemistry is a hybrid science. Biology is the science of living
organisms and Chemistry in the science of the atoms and molecules, so Biochemistry is the
science of the atoms and molecules in living organisms.
Its domain encompasses the entire living world with the unifying interest in the chemical
structures and reactions that occur in living systems.

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The chemical context of life.
Only a small subset of the known elements are found in living systems.
- Living organisms require about 25 chemical elements.
 Carbon (C), oxygen (O), hydrogen (H), and nitrogen (N). These four elements make
up 96% of an organism’s weight.
 Phosphorus (P), sulfur (S), calcium (Ca), potassium (K), and a few other elements
account for the most remaining 4% of an organism’s weight.
 Macromolecules are very large molecules consisting of thousands of atoms.
- Organic molecule: a complex molecule that is primarily made of carbon atoms
bonded with other elements and/or other carbon atoms.
The Functional Groups Most Important in the Processes of Life:
- Hydroxyl group (OH)
- Carbonyl group (C=O)
- Carboxyl group (COOH)
- Amino group (NH2)
- Sulfhydryl group (SH)
- Phosphate group (PO4)

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Cells contain four major types (macromolecules) of biomolecules that are essential for life.
- Proteins.
- Lipids, including fats.
- Nucleic acids (DNA/RNA)
- Carbohydrates.
Most of the macromolecules are large polymers.
Polymers are large molecules consisting of many building units (monomers) linked by
covalent bonds.
The main monomers are:
1- Amino acids: monomers of proteins.
2- Monosaccharaides: monomers of carbohydrates.
3- Nucleotides: monomers of nucleic acids.

The monomers are called residues after they have been incorporated into the polymer.

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Electrotrostatic forces or interactions.
Electrostatic interactions are between and among cations (+ charge) and anions (- charge),
which are species with formal charge of ...-2,-1,+1,+2...
Electrostatic interactions can be either attractive or repulsive, depending on the signs of the
charges. Like charges repel, whereas unlike charges attract. In protein folding, RNA folding
and DNA helix, electrostatic interactions are dependent on salt concentration and pH.
So, in conclusion, electrostatic forces or interactions are between charges, partial or total.

A partial charge is a non-integer charge value when measured in elementary charge units.
It is represented by the greek lowercase 𝛿, namely 𝛿- or 𝛿+.
Partial charges are created due to the asymmetric distribution of electrons in chemical bonds.
It indicates which atom in a bond have a lower electronegativity and which have a higher
electronegativity.

A formal (total) charge indicates gain or loss of electrons while forming covalent bonds.
- Water Molecules Form hydrogen Bonds
The human body, for example, is about 60% by weight water, most of it in the extracellular
fluid (the fluid surrounding cells) and inside cells.
The water molecule is polar.
The polar nature of the water molecule allows water molecules to form hydrogen bonds with
each other and with dissolved hydrophilic (ionic and polar) substances.
- Here polar means: can produce hydrogen bonds.

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 Hydrogen bonds are a type of electrostatic force or interaction.
A hydrogen bond is a relatively weak bond between hydrogen atoms and nitrogen,
oxygen, or fluorine atoms.

- Hydrogen bond in between two partial charges.

Ionic Bonds is an electrostatic interaction between two oppositely charged ions.

- Ionic bond is between a partial charge and a total charge.
-
Example: electrostatic interactions between charged groups such as Na+ and Cl- or
carboxylate (COO-) and amino (NH3+) groups.

van der Waals interactions occur between nonpolar molecules and these forces act
only when the groups are very close. (‫)تحدث بسبب تقارب الكهروسالبية‬

Covalent bonds result when atoms share electrons.

 Double bond is stronger that single bond.

Although hydrogen bonds and van der Waals interactions are individually weak,
biological molecules usually contain multiple groups capable of participating in these
intermolecular interactions, so their cumulative effect can be significant.

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 ‫الكالوري أكبر من الكيلوجول ‪‬‬

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Hydrophobic interaction or effect.
Glucose and other readily hydrated substances are said to be hydrophilic (water loving). In
contrast, a compound such as fat which lacks polar groups, is relatively insoluble in water and
is said to be hydrophobic (water-fearing).

The aggregation of nonpolar groups in water leads to the release of water molecules, initially
interacting with the nonpolar surface, into bulk water. The release of water molecules into
solution makes the aggregation of nonpolar groups favorable.

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Sterochemistry
Isomers are compounds with the same molecular formula but different structures and
properties.
- Cis-trans isomers have the same covalent bonds but differ in spatial arrangements

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Enantiomers are isomers that are mirror images of each other

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 Chapter 2
Amino acids

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  Amino acids
Notes:
1. Chiral: has 4 different single bonds.
2. Tetrahedral: an atom with four attachments, and bond angles of approximately
109.5.
So every chiral is tetrahedral, but not every tetrahedral is chrial.
3. Amino acid is a monomer for protein.

- The charge of the ionized form of amino acids depend on the R group charge.

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 - There is 20 amino acids, 9 of them are essential for human.
1. Valine.
2. Leucine.
3. Lysine.
4. Phenylalanine.
5. Tryptophan
6. Histidine.
7. Isoleucine.
8. Methionine.
9. Threonine.

 Compounds derived from common amino acids.

 Amino acids have two functions:

1- Forming peptide/polypeptide (protein).
2- Forming compounds.

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  Hydrophobic amino acids.

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  Polar amino acids

In addition, the set includes asparagine and glutamine, two amino acids that contain a
terminal carboxamide. The side chain of glutamine is one methylene group longer than that of
asparagine.

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  Positively charged amino acids

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  Negatively charged amino acids

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 Glycine (Gly, G)
 The smallest and simplest amino acid.
 Aliphatic side chain.
 Achiral

 Alanine (Ala, A)
 The second smallest amino acid.
 Chiral
 Aliphatic side chain.

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 Valine (Val, V)
 Chiral.
 Aliphatic side chain.

 Methionine (Met, M)
 Chiral.
 Aliphatic side chain.
 Has a sulfur group.

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 Leucine (Leu, L)
 Chiral.
 Aliphatic side chain.
 Leucine and Isoleucine are structural isomer.

 Isoleucine (Ile, I)
 2 chiral centers.
 Aliphatic side chain.

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 Proline (Pro, P)
 Chiral.
 Aliphatic side chain.
 Proline (Pro) is unique among the amino acids because its aliphatic side chain is also
covalently linked to its amino group.

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  Tryptophan is the biggest amino acid.

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  Cysteine is a thiole, can make a covalent bond.

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  It can make hydrogen bond.
 Aspargine is more polar than glutamine.
 Amides are derived from carboxylic acids.

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  Asparagine is derived from aspartate.
 Glutamine is derived from glutamate.
 The charge is continues/continuum, not all molecules
lose electrons together. That’s why the two carboxyl
groups lose the proton separately.
(‫)المجموعة األقرب على الكربون الوسطي تخسر البروتون أول‬
 The range of charges for acidic amino acids= 1+ - 2-
pH = 0- 3.4 -> 1+
pH = 3.4 -> 0
pH = 3.4 – 7 -> 1-
pH = 8 -> 2-
 Aspartic acid and glutamic acid are amino acids that have
an additional carboxyl group that can release a proton
and acquire a negative charge at the pH of body fluids.
Often, these amino acids are designated with the name of
their ionized form, aspartate and glutamate, respectively.
Note: everything that ends with “ate” is a base.

Both forms here are basic, because the R-group is ionized (coo-)

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 Basic side-chains
 Arginine is the strongest basic amino
acid (‫)األكثر بخال بالتبرع‬.
 Histidine is the weakest basic amino
acid.
 The basic amino acid charge range =
2+ - 1-
pH = 0 – 3.4 -> 2+
pH = 3.4 – 8 -> 1+
pH = 8 – 12 -> 0
pH = 12 -> 1-
 Positive charge will be with the
double bond.
 Negative charge won’t be with the
double bond.
 Histidine can bind or release protons
near physiological pH.

Arginine group

Histidine group
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Protonated (acid) Deprotonated (base)

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 Peptide bonds link amino acids in proteins
O from the carboxyl group and 2H from the amino group.
This reaction is called dehydration reaction.

We notice that the R groups became opposite to each other (Trans), this happens to avoid
magnetic clash/repulsion.

 Tyrosine is the first amino acid. Leucine is the last amino acid.
 The first amino acid is called amino terminal residue.
 The last amino acid is called carboxyl terminal residue.
 The start of the chain is called amino end.
 The end of the chain is called carboxyl end.

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  A polypeptide chain consists of a regularly repeating part, called the main chain or
backbone, and a variable part, comprising the distinctive side chains. The
polypeptide backbone is rich in hydrogen bonding potential.

 Hydrolysis (hydration) of a peptide bond

Happens in the stomach (acidic conditions).
It’s hard to hydrolysis the protein, that’s why we need strong acidic conditions
(environment).
If we want to mimic the hydrolysis outside the stomach, we need heat instead of enzymes,
and the process will take a long time (almost a full day).
 Peptide bonds can be broken, or hydrolyzed, by the action of exo- (e.g. carboxypeptidase)
or endopeptidases (e.g. Trypsin, pepsin) (enzymes that act from the end or the middle of
the chain, respectively).

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 Functional Significance of Amino Acid R-Groups
The hydrophobic amino acids will generally be encountered in the interior of proteins
shielded from direct contact with water.

Conversely, the hydrophilic amino acids are generally found on the exterior of proteins as
well as in the active centers of enzymes. Indeed, it is the very nature of certain amino acid
R-groups that allow enzyme reactions to occur.

The imidazole ring of histidine allows it to act as

either a proton donor or acceptor at physiological
pH. Hence, it is frequently found in the reactive
center of enzymes.

The formation of disulfide bonds (a covalent bond) between cysteines present within
proteins is important to the formation of active structural domains (active sites in the
protein) in a large number of proteins. 

Outside the cell (extracellular space)

Inside the cell

2 Chains, 3 disulfide bonds

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 Optical Properties of the Amino Acids
A tetrahedral carbon atom with four different groups or atoms is said to be chiral. The one
amino acid not exhibiting chirality is glycine since it’s '"R-group" is a hydrogen atom.

All of the amino acids in proteins exhibit the same absolute steric configuration and are all
L-a-amino acids. D-amino acids are never found in proteins, although they exist in nature.
D-amino acids are often found in polypetide antibiotics.

The aromatic R-groups in amino acids absorb ultraviolet light with an absorbance maximum
in the range of 280nm. The ability of proteins to absorb ultraviolet light is predominantly
due to the presence of the tryptophan, which strongly absorbs ultraviolet light.

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 Acid-Base Properties of the Amino Acids
Relationship between [H+] and [OH-]
* Water ionizes to form H+ and OH-

The products of water’s dissociation are a hydrogen ion or proton (H+) and a hydroxide ion
(OH-).
Other examples:

The concentration of protons [H+] in aqueous solution is expressed as pH, where pH = – log
[H+]
The concentration of a hydroxide ion [OH-] in aqueous solution is expressed as pOH, where
pOH = -log [OH-]

Water ionization:

Take –log of both sides for convenience:

* Therefore, the pH scale ranges from 0 to 14

A neutral solution has a pH of 7, an acidic solution has a pH
< 7, and a basic solution has a pH > 7.
The so-called physiological pH, the normal pH of human
blood, is a near-neutral 7.4.

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 The carboxyl group as acidic group (-COOH) and amino group as a basic group (-NH2) groups
in amino acids are capable of ionizing.

The carboxylic acid deprotonate on lower pH values <7 (lower than 7), the amino group on
the other hand, deprotonate on higher pH values >7 (higher than 7).
AA has both ACID and BASE groups in same molecule; an amino acid can act as a buffer
because it can react with added acids and bases to keep the pH nearly constant as a buffer.

 Buffer is a solution resists changes in pH upon addition of acid or base

 The pH of a solution of acid is related to the pK

Adding an acid increases the concentration of [H+] and decreases the pH; adding a base has
the opposite effect. Biochemists define an acid as a substance that can donate a proton
and a base as a substance that can accept a proton.

Weak Acid Conjugate base Proton

The final pH depends on the equilibrium between HA and A-

Ka = [H+][ A-]/ [HA] or [H+] = Ka [HA] /[A-]
Ka is the acid dissociation constant
* Equation is known as the Henderson–Hasselbalch equation. It relates the pH
of a solution to the pK of an acid and the concentration of the acid (HA) and its
conjugate base (A-).

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 Henderson – Hasselbalch equation.

Therefore, when the concentration of conjugate base and acid are the same, then pH is equal
to pKa.

- Every amino acid has at least two pK

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  Amino acids Ionization Acid Base

Buffer #1
‫ثنائي التأين‬
pK1 pK2 Buffer #2

Buffer solution forms when we have a mixture of base with its conjugate acid or acid with its
conjugate base.

When pH levels rise, COOH/NH3+ start to lose “H”.

At pH = 2, pK1 (alpha-carboxyl) = pH
At pH = 9, pK2 (alpha-amino) = pH
Has the ability to act as base or acid.
When near the “red”, it is a conjugate base.
When near the “green”, it is a conjugate acid.
It is considered amphoteric: a molecule or ion that can react both as an acid and as a base.
- The predominant form of the amino acid present in a solution depends on the pH of the
solution and on the nature of the amino acid. In strongly acidic solutions all amino acids are
present primarily as cations; in strongly basic solutions they are present as anions.

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 - +
R-COOH  R-COO + H
+ +
R-NH3  R-NH2 + H
- The isoelectric point (pI) is the pH value when the net charge of the amino acid
(protein) is zero.
pK1+pK2
- 𝑝𝐻 =
2

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  pKa values (in water)
- Only 7 amino acids have R groups which can be ionized.
- Cysteine and Tyrosine are special, because they are not basic nor acidic, but still can
be ionized. Like the acid, they get deprotonated on high pH values.

- Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R-
groups in amino acids can be defined by the dissociation constant, Ka or more
commonly the negative logrithm of Ka, the pKa
- Ka = [H+][ A-]/ [HA]
pH = pKa + log [A-]/ [HA]
- pKa values depend on temperature, ionic strength, and the microenvironment (R-
group) of the ionizable group.

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  Titration curve for Alanine

Moles

1- When adding 0.5 moles of OH, COOH halfway deprotonate into COO-.
2- When adding 1 moles of OH, COOH fully deprotonate into COO-.
3- When adding 1.5 moles of OH, NH3+ halfway deprotonate into NH2.
4- When adding 2 moles of OH, NH3+ fully deprotonate into NH2.

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  pKa values of amino acids.
The higher the pKa, the weaker the acid, and the stronger the base.

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 Chapter 3
Protein Structure and Function

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  Protein Structure and Function
Proteins make up 50% of the dry weight of most cells. In most cases, a protein's
function depends on its complex three-dimensional structure.
Each protein molecule consists of one or more chains of amino acid monomers.
A single chain is named polypeptide.
There are two major conformations of proteins: Fibrous (‫ليفي‬/‫ )خطي‬proteins and globular
proteins (‫)كروي‬

1. globular proteins:
Usually water soluble, compact (filled from the inside), roughly spherical
Hydrophobic interior, hydrophilic surface.
Globular proteins include enzymes, carrier (hemoglobin) and regulatory proteins.

- The protein comprised a hydrophilic surface and hydrophobic core especially in an

aqueous solution. ‫إذا كنا في بيئة زيتية ينعكس الوضع‬

2. Fibrous protein:
Provide mechanical support.
Often assembled into large cables or threads.
Fibrous proteins can be subdivided into several different types:
1- Keratins, found in hair, fingernails, and bird feathers
2- Collagens – the most abundant proteins in a vertebrate body – found in
connective tissues such as cartilage
3- Elastins, found in ligaments, around blood vessels.

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  Important fibrous proteins
1. Intermediate filaments of the cytoskeleton
Structural scaffold inside the cell.
Keratin in hair, horns and nails.
2. Extracellular matrix
Bind cells together to make tissues.
Secreted from cells and assemble in long fibers.
- Collagen – fiber with a glycine every third amino acid in the protein.
- Elastin – unstructured fibers that gives tissue an elastic characteristic.

Collagen and Elastin

3 chains

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  Protein structure
There are as many as four levels of structure in a protein.
1. Primary structure – Sequence of amino acids that comes from peptide bond
formation.
2. Secondary structure – α-helix, β-strand, turn that comes from hydrogen bonds
(NH - C=O) in the backbone.
3. Tertiary structure (fold), one chain, this comes from R-group interactions.
These interactions can be:
- Polar: H-bond.
- Acidic vs Basic: ionic bond.
- Nonpolar: Van der Waals interaction.
- Cysteine with Cysteine: disulfide – Covalent.
4. Quaternary structure – Subunits, more than one chain.
Interactions responsible for this structure are R-group interactions.

Tertiary structure interactions are between the same peptide chain.

Quaternary structure interactions are between different peptide chains.

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The sequence of amino acids in a polypeptide is called the protein’s primary structure. (i.e. the
primary structure is like the order of letters in a very long word)
There are so many possible primary structures, so many ways the twenty amino acids can be
joined, so the number of proteins that can be made is almost limitless.
Different amino acids have different properties, so primary structure shapes the
three dimensional folding of the protein. It is the folding of the protein that
determines its function.

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  Under physiological conditions, a polypeptide folds up to form a more compact shape
Secondary structure regions stabilized mainly by hydrogen bonds between atoms of
the polypeptide backbone (not the amino acid side chains).

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 There are two types of secondary structure:
1. α-helix Ball and stick model

Backbone
R-group

Space

filling

model.

- The amino acid “i” always binds with amino acid “i+4”.
- “i” gives C=O and “i+4” gives NH.
- Space filling model: a graphic or physical representation of a molecule in
which the atoms are partial spheres that have diameters proportional to
those of the real atoms and that are joined directly to one another
- Ball and stick model: three-dimensional models where atoms are represented
by spheres of different colors and bonds are represented by sticks between
the spheres.
- The key difference between ball and stick and space filling model is that in the
ball and stick model, the molecular structures are depicted by spheres and
rods whereas, in the space-filling model, the molecular structures are
depicted by full-sized spheres without rods.
- The height of a single turn is 5.4Å
- The height of a single amino acid in turn is 1.5 Å
- So how many amino acids we have in one turn? 3.6 residues/turn
- It’s logical for all amino acids to have the same height, because R-groups
didn’t contribute to this structure, only the backbone.
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 - Not all amino acids favor the formation of the α-helix due to steric constraints
of the R-groups.
1. Amino acids such as Ala, Asp, Glu, Ile, Leu and Met favor the
formation of α-helices
2. Whereas, Gly and Pro favor disruption of the helix. This is particularly
true for Pro since it is a pyrrolidine based amino acid (HN=) whose
structure significantly restricts movement about the peptide bond in
which it is present, thereby, interfering with extension of the helix.
The disruption of the helix is important as it introduces additional
folding of the polypeptide backbone to allow the formation of
globular proteins.
 α-helix stabilization
Is stabilized by hydrogen bonds, between the carboxyl O of the i residue and the
H of the amid of the i+4 residue

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 In the α helix, the carbonyl oxygen of each residue forms a hydrogen bond with
the backbone NH group four residues ahead.

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 2. β-strands

Same chain that has a turn

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As evident from the above diagram, parallel beta sheets are less stable than anti-parallel beta
sheets, because the geometry of the individual amino acid molecules forces the inter chain
hydrogen bonds in parallel beta-pleated sheets to occur at an angle, making them longer and
thus weaker than those in anti-parallel beta-pleated sheets, where the inter strand hydrogen
bonds are aligned directly opposite each other, resulting in stronger and more stable bonds.

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 - Turns reverse the direction of the protein.

Omega loops can contribute to protein

function. For example, omega loops can help
stabilize interactions between protein and
ligand.
Loop ‫سطح االرتباط دائما يكون‬
Omega loop does not change depending on
the organism, the human, or the animal

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 Tertiary structure
Tertiary structure is the overall shape of a polypeptide resulting from interactions
between the side chains (R groups) of the various amino acids.
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units of a given protein.
The tertiary structure is the structure at which polypeptide chains become functional.

‫حديد واحدة‬
‫له القدرة على نقل جزيء اكسجين واحد‬

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Distribution of amino acids in myoglobin

Examples of folded protein

A protein that stimulates the

growth of new blood vessels.

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Domain: is a polypeptide segment that has folded into a single structural unit.
Domain is a supersocondary structure that is not a secondary structure nor tertiary structure,
it’s something in between.
Tertiary structure consists of multiple domains.

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 Quaternary structure
- Proteins containing more than one polypeptide chain have quaternary structure.
- The individual chains, called subunits.
- The forces that hold subunits together are similar to those that determine the
tertiary structures of the individual polypeptides.
- Proteins with multiple polypeptide chains are termed oligomeric proteins.
- Oligomeric proteins can be composed of multiple identical polypeptide chains or
multiple distinct polypeptide chains. Proteins with identical subunits are termed
homooligomers.

Proteins containing several distinct polypeptide chains are termed

heterooligomers.

- Hemoglobin, the oxygen carrying protein of the blood, contains two α and two β
subunits arranged with a quaternary structure in the form, α2β2. Hemoglobin is,
therefore, a hetero-oligomeric protein.

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A picture from Campbell book

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Disulfide bonds, a covalent bond stabilizes the tertiary structure.

First, the tertiary structure is formed, and then the disulfide bonds (covalent bonds) occur.
- Denaturation and Renaturation of proteins.
Denaturing agent can break all bonds except disulfide bonds, and peptide bonds.
Reducing agent can breaks the disulfide bond.

Reducing agent

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Protein Denaturation
Peptide bonds are not broken

Denaturing agents

Urea did the denaturation of the native ribonuclease

Β-mercaptoethanol did the reduction of the native ribonuclease.
The process of denaturation takes the protein back to its primary structure.
Since denaturation reactions are not strong enough to break the peptide
bonds, the primary structure (sequence of amino acids) remains the same
after a denaturation process.
Denaturation occurs because the bonding interactions responsible for the
secondary structure (hydrogen bonds to amides) and tertiary structure are
disrupted.
A variety of reagents and conditions can cause denaturation, such as
1. Heat
2. Organic solvents
3. pH
4. Salt

Alfred E. Mirsky, The discoverer of denaturation process.

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Renaturation (Denaturation ‫)عكس عملية‬ To get the protein back to its normal
state, we remove the denaturation agent
first (here urea), then we remove the
reduction agent (β-mercaptoethanol)

No amino acid sequence can fold to another sequence. Each sequence has a specific
structure.

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 Protein post-translational modifications.
Not all proteins have post-translational modifications.
When protein is covalently conjugated with a molecule other than amino acid.
Examples:
Glycation => Glycoprotein (There are at least 100 blood group determinants, most of
which are due to carbohydrate differences).
There are extremely important glycoproteins found on the surface of erythrocytes. It is
the variability in the composition of the carbohydrate portions of many glycoproteins
and glycolipids of erythrocytes that determines blood group specificities.

Phosphorylation: addition of phosphate group.

Lipoproteins: (storage & transport of lipid and cholesterol)

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 Cells have a determined lifetime
For example, Erythrocytes production in a 70 Kg man:
Erythrocytes concentration: 5 x 109 cells/ml
Blood volume: 5 Lit
Total cell number: 5000 ml x 5 x 109 cells/ml = 2.5 x 1013cells
Lifetime of an Erythrocyte: 120 days

2.5 x 1013 cells/120 days = 2.0 x 1011 Erythrocytes per day

2.0 x 1011 cells/24 hours = 8.6 x 109 Erythrocytes per hour
8.6 x 109 cells/60 minute = 1.4 x 108 Erythrocytes per minute
1.4 x 108 cells /60 second = 2.4 x 106 Erythrocytes per second
Hemoglobin about 30 picograms/cell
2 x1011 Erythrocytes/day x 30 picograms/cell =
6x1012 picograms hemoglobin/day = 6g hemog./day

 Protein cellular function

1. Transport and storage, for example, hemoglobin (transport), albumin (storage).
2. Movement and contraction, for example, actin and myosin.
3. Immune system antibodies.
4. Nerve signaling, nerve growth factors, for example, neutrophin.
5. Differentiation DNA and histone modifiers.
6. Blood clotting, for example, fibrin.
7. Cells and organ structure.
8. Enzymes, hormones, for example, insulin.
Enzymes, enzymes speed up chemical reactions, examples: amylase and proteases
Hormones, such as insulin
Receptors, such as G-protein receptor
Transport proteins, examples: hemoglobin and cell membrane proteins
Structural proteins, examples: elastin, collagen, and keratin
Storage proteins, examples: ovalbumin, casein, ferretin
Gene regulation, such as histones
Immune protection, antibodies, examples: Ig.E, IgA, and Ig.G
Coordinated motion and contractile movement, examples: actin and myosin
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 Chapter 4
Enzymes

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 Enzymes
Living systems use catalysts called enzymes to increase the rates of chemical reactions.
Enzymes are biological catalysts responsible for supporting almost all of the chemical
reactions that maintain animal homeostasis.
 Homeostasis is the maintenance of a constant (yet also dynamic) internal
environment in terms of temperature, pH, and water concentrations, etc...
Enzymes are proteins and certain class of RNA (ribozymes) which enhance the rate of a
thermodynamically feasible reaction and are not permanently altered in the process.

Many drugs are enzyme inhibitors.

Lipitor: HMG-CoA reductase, inhibits a liver enzyme that is important in biosynthesis of
cholesterol (>$100 billion total sales since 1996).
Ibuprofen: inhibits the cyclooxygenase enzymes.
Saquinavir: protease inhibitor, anti-retrovirus (HIV).

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 Enzymes are found in all tissues and fluids of the body:
1. Intracellular enzymes catalyze the reactions of metabolic pathways.
2. Plasma membrane enzymes regulate catalysis within cells in response to
extracellular signals.
3. Enzymes of the circulatory system are responsible for regulating the clotting of
blood.
 Almost every significant life process is dependent on enzyme activity:
1. Enzymes increase the reaction rate as much as 1017 fold.
2. Enzymes operate in moderate temperature and neutral pH (Physiological
conditions).
3. Most enzymes are absolute or near-absolute specific to the substrates.
 Enzymes speed up reactions by lowering activation energy. The lower the activation
energy for a reaction, the faster the rate.

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 Nomenclature of Enzymes
Trivial name
Gives no idea of source, function or reaction catalyzed by the enzyme. Example: trypsin,
thrombin, pepsin.
Systemic name
The enzyme's name is comprised of the names of the substrate(s), the product(s) and
the enzyme's functional class.
According to the International union Of Biochemistry, an enzyme name has two parts:
1. First part is the name of the substrates for the enzyme.
2. Second part is the type of reaction catalyzed by the enzyme.This part ends with
the suffix “ase”. Example: Lactate dehydrogenase

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 EC number
- Enzymes are classified into six different groups according to the reaction being
catalyzed.
- The nomenclature was determined by the Enzyme Commission in 1961 (with the
latest update having occurred in 1992), hence all enzymes are assigned an “EC”
number.
- The classification does not take into account amino acid sequence (ie, homology),
protein structure, or chemical mechanism.
- The classification depends on the chemical reaction.
- EC number is used to enter enzymes as data.
EC numbers are four digits, for example a.b.c.d:
“a” is the class, 6 classes.
“b” and “c” digits describe the reaction.
“d” distinguishes between different enzymes of the same function based on the actual
substrate in the reaction.
Example: for Alcohol: NAD+oxidoreductase EC number is 1.1.1.1
The first enzyme that was classified.

For example: Isopropyl malate dehydrogenase: EC 1.1.1.85

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 1. Oxidoreductase
EC 1. Oxidoreductases: catalyze the transfer of hydrogen or oxygen atoms or electrons
from one substrate to another, also called oxidases, dehydrogenases, or reductases.
Note that since these are ‘redox’ reactions, an electron donor/acceptor is also required
to complete the reaction.
2. Transferases (‫)النقل يكون من الخارج‬
EC 2. Transferases: catalyze group transfer reactions, excluding oxidoreductases
(which transfer hydrogen or oxygen and are EC 1). These are of the general form:
A-X + B ↔ BX + A
3. Hydrolases (‫)تحليل بالماء‬
EC 3. Hydrolases: catalyze hydrolytic reactions. Includes lipases, esterases, nitrilases,
peptidases/proteases. These are of the general form:
A-X + H2O ↔ X-OH + HA
4. Lyases (‫)تحليل بدون ماء‬
EC 4. Lyases: catalyze non-hydrolytic removal of functional groups from substrates,
often creating a double bond in the product; or the reverse reaction, ie, addition of
function groups across a double bond.
A-B → A=B + X-Y
X Y
Includes decarboxylases and aldolases in the removal direction, and synthases in the
addition direction.
5. Isomerases (‫)النقل يكون من داخل الجزيء نفسه‬
EC 5. Isomerases: catalyze isomerization reactions, including racemizations and cis-tran
isomerizations.
Intramolecular group transfer.
6. Ligases
EC 6. Ligases: catalyzes the synthesis of various (mostly C-X) bonds, coupled with the
breakdown of energy-containing substrates, usually ATP.

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Enzymes are also classified on the basis of their composition. Enzymes composed wholly of
protein are known as simple enzymes in contrast to complex enzymes, which are composed
of protein plus a relatively small organic molecule. Complex enzymes are also known as
holoenzymes.
- In this terminology the protein component is known as the apoenzyme, while the
non-protein component is known as the cofacter
- Metalloenzymes: Enzymes that have a Me+ as a cofactor.
- Coenzyme, when the organic molecule is bound to the apoenzyme by non-covalent
bonds.
- Prosthetic group, when the organic molecule is bound to the apoenzyme by
covalent bonds.
- Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins
- Cofactor can be:
1. A coenzyme - a non-protein organic substance which is loosely attached to
the protein part.
2. A prosthetic group – a non-protein organic substance which is firmly
attached to the protein or apoenzyme portion.
3. A metal-ion-activator - these include K ,+Fe ,++Fe ,++Zn ,++Mg ,++Ca

Apoenzyme + Cofactor = Holoenzyme

Non-
covalent
bond

Covalent bond
Ionic bond

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 Cofactors

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 - Wanted: memorize each coenzyme and its vitamin

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 Specificity of Enzymes
One of the properties of enzymes that makes them so important as diagnostic and
research tools is the specificity they exhibit relative to the reactions they catalyze, other
enzymes will be specific for a particular type of chemical bond or functional group:
In general, there are four distinct types of specificity:
1. Absolute specificity - the enzyme will catalyze only one reaction.
For example, Succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction
reaction and its substrate is invariably succinic acid.
2. Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
For example, alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction
reactions but attacks a number of different alcohols, ranging from methanol to
butanol. Generally, enzymes having broad substrate specificity are most active
against one particular substrate. In the case of ADH, ethanol is the preferred
substrate
3. Linkage specificity - the enzyme will act on a particular type of chemical bond,
regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or optical
isomer. Enzymes that attack D sugars will not attack the corresponding L isomer.
Enzymes that act on L amino acids will not employ the corresponding D optical
isomer as a substrate.
 Isozymes: The products of genes that vary only slightly (10-15%); often, various isozymes
of a group are expressed in
different tissues of the body.
Takes the “same” Substrate
and produces the same
Product.
Isozymes have the same
function but differ in the speed
depending on the tissue it is
expressed in.

Isozymes have the number of

2.7.1.d
They differ in the number “d”

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  Enzyme-Substrate interactions
S + E ↔ ES P + E
Enzymes are catalysts and increase the speed of a chemical reaction without themselves
undergoing any permanent chemical change. They are neither used up in the reaction nor do
they appear as reaction products.
E: The enzyme catalyzing the reaction,
S: The substrate, the substance being changed
P: The product of the reaction
K1 depends on the diffusion.
K1- depends on the strength of the bond between S and E (affinity), if the bond is weak, it’ll
dissociate.
K2 depends on the catalyst.

K1

K1-

At this point, the complex can dissociate

‫خطوة انعكاسية‬

K2

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1 unit (U) is the enzyme activity that converts 1μmole of substrate per min under standard
conditions.
The specific activity of an enzyme is defined as the activity per unit of mass or U/mg protein.
E Catalysts speed up the forward and reverse reactions proportionately (the equilibrium
constant remains the same in the presence or absence of enzyme).
E have no effect on the equilibrium constant of the reactions they catalyze.

 E reduces ∆G‡ for Activation

∆G < 0

∆G is the free energy change for the reaction.

When ∆G for the reaction is negative so the reaction proceeds spontaneously
When ∆G for the reaction is positive so the reaction does not proceed spontaneously
The energy-requiring step of the reaction is shown as an energy barrier, called the free
energy of activation or activation energy and symbolized ∆G ‡.

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 - Exergonic happens by itself.
- An endergonic reaction occurs by coupling with an even more exergonic reaction.
- ‫طاقة التفاعل الكلية لم تتأثر‬
- ‫الناتج األخير لم يتأثر‬
- ‫الشيء الوحيد الذي تأثر هو طاقة التنشيط‬

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Models of Enzyme-Substrate Binding
 Lock and key model:
This means that the enzyme’s tertiary structure consists of a unique pocket or site, which
is tailor-made to fit only its substrate and nothing else, just as a key fits into a lock.
(Explained above – Page 83).
 Induced-fit model:
This updated model states that enzymes interact with substrates and in the process
change their conformation such that the enzyme is snug around the substrate, sort of like
a glove around a hand.

Both the enzyme and substrate change its shape to fit with each other.

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 Factors affecting reaction rates
The study of enzyme reaction rates is called enzyme kinetics. Enzyme kinetics is affected
by:
1. Temperature and pH: Each enzymatic reaction has an optimum pH and optimum
temperature. Extreme temperature or pH disrupts enzyme structure and therefore
reaction rate.
Changing the pH of its surroundings will also change the shape of the active site of
an enzyme.
Increasing the temperature increases the velocity and kinetic energy.

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 2. Enzyme concentration
At saturating substrate concentration, the initial rate is directly related to the
enzyme concentration.
E + S = ES = E + P
Thus, as long as S is not limiting, more E leads to more ES.

The shape of the reaction

velocity versus enzyme
concentration curve is linear

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 3. Substrate concentration
The reaction rate can be increased by adding more substrate, or by removing
product as it is formed.

When the enzyme concentration

is held constant, the reaction
velocity varies with the substrate
concentration, but in a nonlinear
fashion (hyperbolic)

As the substrate concentration

increases the reaction rate does
the same, because there is more
substrate for the enzyme to react
with.

- Reaction rates (reaction velocities): To measure a reaction rate, we monitor the

disappearances of reactants or appearances of products.
V=∆P/∆t
- initial velocity => [product] = 0, no back reaction

Progress of the triose phosphate isomerase

reaction. Over time, the concentration of the
substrate glyceraldehyde-3-phosphate
decreases and the concentration of the
product dihydroxyacetone phosphate
increases.

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For example, NO as a product.

V=∆P/∆t= (P2-P1)/(t2-t1)  V0 (initial): P1=0; t1=0

We take the average rate not the instantaneous rate. Because the instantaneous rate is
derivative.
 Reaction order
- Zero Order: the rate of a zero-order reaction is independent on the concentration of
the reactant(s). Zero-order kinetics are observed when an enzyme is saturated by
reactants. At saturation, we reach the maximal velocity.
The output is maximum at saturation point, after the saturation point, the output
does not increase.

At high substrate concentration, the reaction rate reaches a plateau (the maximum
point) as the enzyme active sites become saturated with substrate (ES complex), and
no free enzyme to bind with the added substrate.

- First Order: the rate of a first-order reaction varies linearly on the concentration of
one reactant. First-order kinetics are observed when a protein folds and RNA folds
(assuming no association or aggregation).
The reaction begins as first order then becomes zero order.

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 Kinetics is the study of reaction rates (time-dependent phenomena).
We will study kinetics through substrate concentration.
 Rates of reactions are affected by
1. Enzymes/catalysts.
2. Substrates.
3. Effectors (inhibitors or activators).
4. Temperature.
5. Concentrations.
When studying kinetics, all of these factors will be constant except for substrate
concentration.

 Why study enzyme kinetics?

- Quantitative description of biocatalysis.
To understand how the drug work in the body.
In addition, understand some pathogenic mechanisms.
- Understand catalytic mechanism.
- Find effective inhibitors.
- Understand regulation of activity.

 General observation
- Enzymes are able to exert their influence at very low concentrations ~ [enzyme] = nM.
We know from before that enzyme concentration is low in the body.
- The initial rate (velocity) is linear with [enzyme].
- The initial velocity increases with [substrate] at low [substrate].
- The initial velocity approaches a maximum at high [substrate].

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Both Vmax and kM are constant, when enzyme
concentration, pH and temperature are constant.
However, changing the temperature will affect the
kM, and changing the enzyme concentration will
affect Vmax.

kM (Michaelis-Menton constant): the substrate concentration when velocity is equal to half

of Vmax (kM=Vmax/2).

KM has the same unit as [S], usually Molarity.

Vmax: the maximal velocity reached when saturation occurs.

We reach Vmax/2 when Km and [S] are equal.
Example: Assume than [S] = 5 and Km = 5
𝑽𝒎𝒂𝒙∗𝟓 𝟓𝑽𝒎𝒂𝒙
V= = = Vmax/2
𝟓+𝟓 𝟏𝟎 2
We reach Vmax when [S] is much higher than KM, so we ignore KM value.

V = Vmax[S]/{Km +[S]} -> Michaelis-Menten equation

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  Michaelis-Menton Kinetics
Simplest enzyme mechanism
- One reactant (S)
- One intermediate (ES)
- One product (P)

- In typical enzyme-catalyzed reactions, substrate and product concentrations are

usually hundreds or thousands of times greater than the enzyme concentration.
Consequently, each enzyme molecule catalyzes the conversion to product of many
reactant molecules.
- The catalytic event that converts substrate to product involves the formation of a
transition state, and it occurs most easily at a specific binding site on the enzyme.
- This site, called the active site of the enzyme.
- The enzyme binds quickly non-covalently to the substrate to form a non-covalent ES
complex (ES)
 The ES complex is known as the Michaelis complex.
 A Michaelis complex is stabilized by molecular interactions (non-covalent
interactions)
 Michaelis complexes form quickly and dissociate quickly.

- ES complex undergoes a chemical transformation and dissociate to give product (P)

and enzyme (E) - enzyme product complex (EP), and this step is very slow.
- The series of events can be shown thus:

V=k2[ES] (second step is the rate determining step)

- The kinetic was first characterized by biochemists Michaelis and Menten.

- Many enzymatic reactions follow Michaelis–Menten kinetics, even though enzyme
mechanisms are always more complicated than the Michaelis–Menten model.
- For real enzymatic reactions use kcat instead of k2.
- k1 and k-1 are usually much faster than kcat.

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  Kcat and the reaction velocity

- The enzyme is either free ([E]) or bound ([ES]): [Eo] = [ES] + [E].
- At sufficiently high [S], all of the enzyme is tied up as ES (i.e., [Eo] ≈ [ES])
- At high [S], the enzyme is working at full capacity (V = Vmax).
- The full capacity velocity is determined only by Kcat and [Eo].
- kcat = turnover number: number of moles of product produced per time per enzyme
active site.
The higher the kcat the better the enzyme.

 For any enzyme it is possible (pretty easy) to determine kcat.

 To understand and compare enzymes we need to know how well the enzyme binds to S
(what happens in the first part of the reaction.) kcat does not tell us anything about how
well the enzyme binds to the substrate.

 Assumptions.
1. k1,k-1>>k2 (the first step is fast and is always at equilibrium).
2. d[ES]/dt ≈ 0 (the system is at steady state)

𝒅[𝑬𝑺]
= rate of formation of ES – rate of breakdown of ES ≈ 0 (at steady state)
𝒅𝒕
Steady state occurs when the rate of formation and breakdown of the intermediate
are equal.
3. There is a single reaction/dissociation step (k2=kcat).
4. There is no back reaction of P to ES. This assumption allows us to ignore
k-2. We start to measure initial velocities, when [P] ≈ 0.

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 )

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  Significance of KM

- kM = [E][S]/[ES] and kM = (k1 + k2)/k1.

- KM is the apparent dissociation constant of the ES complex (when k2<<k-1 ).
A dissociation constant (kD) is the reciprocal (‫ )معاكس‬of the equilibrium constant
(kD=kA-1). KM is a measure of a substrate’s affinity for the enzyme (but it is the
reciprocal (‫ )معاكس‬of the affinity).
KM ‫تعبر عن التفكك وليس االرتباط‬

- The lower the kM the better for the enzyme. We can reach Vmax /2 faster.
- The higher the dissociation the higher kM.
- If k1,k-1>>k2, the kM=kD.

- kM is the substrate concentration required to reach half-maximal velocity (Vmax/2). A

small kM means the substrate binds tightly to the enzyme and saturates (max’s out)
the enzyme.
- The microscopic meaning of kM depends on the details of the mechanism.

 Significance of kcat

- Vmax = kcat [Eo]

- kcat: For the simplest possible mechanism, where ES is the only intermediate, and
dissociation is fast, then kcat=k2.
- kcat is the “turnover number”: indicates the rate at which the enzyme turns over, i.e.,
how many substrate molecules one catalytic site converts to product per second.
- If there are multiple catalytic steps (e.g., trypsin) then each of those rate constants
contributes to kcat.
- The microscopic meaning of kcat depends on the details of the mechanism.
- The higher kcat the better for the enzyme.

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  Significance of kcat/kM

- kcat/kM is the catalytic efficiency. It is used to rank enzymes (which is best and which
is worst).
A big kcat/KM means that an enzyme binds tightly to a substrate (small KM), with a fast
reaction of the ES complex.
Big value -> better enzyme
Small value -> worse enzyme

- kcat/kM can be used to estimate the reaction velocity from the total enzyme
concentration ([E0]).
Max kcat/kM =109/s.M => diffusion control.

kcat/(k-1 + kcat) equals less than one.

kcat/k-1 + kcat * k1 will be less than k1.

- kcat/kM is the specificity constant. It is used to distinguish and describe various

substrates.

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  Data analysis
- It would be useful to have a linear plot of the MM equation
- Lineweaver and Burk (1934) proposed the following: take the reciprocal of both sides
and rearrange.
- Collect data at a fixed [E0].

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  Enzyme inhibition
Enzyme inhibitors fall into two broad classes:
1. Reversible inhibitors -> usually non-covalent.
2. Irreversible inhibitors -> usually covalent.

- Reversible inhibition.
Many drugs are reversible inhibitors
1. Lipitor: HMG-CoA reductase, inhibits a liver enzyme that is important in
biosynthesis of cholesterol
2. Ibuprofen: inhibits the cyclooxygenase enzymes.
3. Saquinavir: protease inhibitor, anti-retrovirus (HIV).
Enzyme reversible inhibitors
Competitive inhibitors.
Compete with substrate for binding to enzyme

- The enzyme either binds to a substrate and form substrate-enzyme complex and
produce the product, or binds to an inhibitor and form inhibitor-enzyme complex
and produce no product.
This process depends on the concentration of the substrate and inhibitor, the one that
has a higher concentration binds to the enzyme.

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Vmax is not changed, Km is increased with inhibition.
Increasing [S] can reverse the inhibition.
The lower the kI the stronger the inhibitor, and vice versa, the higher the kI the weaker the
inhibitor.
The smaller the kI, the greater the binding affinity between inhibitor and enzyme, and the
smaller amount of medication needed in order to inhibit the activity of the enzyme.

kM is reversely
proportional to kI.
kM is directly proportional
to inhibitor strength

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 The stronger
inhibitor

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 Enzyme Reversible inhibitors
Uncompetitive Inhibitors

- Comes after the binding of substrate and it’ll inhibit the activation of the enzyme.
Binds outside the active site and it could affect the active site of the enzyme.

KI prime, same relations as KI for

the ES.

[ ES ][ I ]
 KI ' inhibitor dissociation constant
[ ESI ]

[ E ]0  [ E ]  [ ES ]  [ ESI ] total enzyme concentration

1 KM '
 +
v v max [ S ] v max

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Increasing [S] cannot reverse inhibition
This will result in the reduction of Vmax because the enzymes ability for catalysis is being
reduced by the binding of inhibitor to the enzyme-substrate complex.
kM is reduced by α’ value.

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 Enzyme Reversible inhibitors
Noncompetitive Inhibitors

- Binds to the enzyme without the need of the substrate to bind. (Binds at any context)
- It’ll change the shape of the active site of the substrate and prevent the binding of
the substrate.
- The noncompetitive inhibitor doesn’t bind to the active site of the enzyme
- Noncompetitive inhibition is a mix between competitive and uncompetitive.

Mixed (competitive and uncompetitive) Inhibition

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Increasing [S] cannot reverse inhibition.
kM is unaffected (may increase or decrease slightly).
Vmax is reduced.

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 - The main property of all the reversible inhibitors is that when the inhibitor
concentration drops, enzyme activity is regenerated.

- Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor
maintains a reversible equilibrium with the enzyme.

- The best-known reversible inhibitors are competitive inhibitors, which always bind at
the catalytic or active site of the enzyme. Most drugs that alter enzyme activity are of
this type.

- Competitive inhibitors are especially attractive as clinical modulators of enzyme

activity because they offer two routes for the reversal of enzyme inhibition:

1. Decreasing concentration of the inhibitor reverses the equilibrium of the

reaction and regenerates active free enzyme.

2. Increasing concentration of the substrate (S), while holding the concentration of

inhibitor constant, provides the second route for reversal of competitive
inhibition. The greater the proportion of substrate, the greater the proportion
of enzyme present in competent ES complexes.

- Competitive inhibitor drugs are the best for chronic diseases.

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 - Irreversible inhibition
Inhibitors of this class usually cause an inactivating, covalent modification of enzyme
structure.
 Cyanide is a classic example of an irreversible enzyme inhibitor: by covalently
binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated
with electron transport.
 The kinetic effect of irreversible inhibitors is to decrease the concentration of
active enzyme; the reduction of enzyme concentration will lead to decreased
reaction rates.
 Irreversible inhibitors are usually considered poisons and are generally unsuitable
for therapeutic purposes.

 Penicillin makes a covalent bond with the OH at the active site of the E, a
Glycopeptide transpeptidase (substrate), which builds the peptidoglycan of the
bacterial cell wall.
Penicillin mimics the D-Ala in peptidoglycan.

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  Regulation of enzyme activity

A. Altering the enzyme concentration

Three principal mechanisms are known to regulate the concentration of active
enzyme in tissues:
1. Regulation of gene expression controls the quantity and rate of enzyme
synthesis. (Slow -> hours to weeks)

2. Proteolytic enzyme activity determines the rate of enzyme degradation.

(Faster than 1)

3. Covalent modification (phosphorylation or de-phosphorylation) of

preexisting pools of inactive proenzymes produces active enzymes. (The
fastest way)
This process is reversible.
Proenzymes are generally synthesized in abundance, stored in secretory
granules and covalently activated upon release from their storage sites.
Examples of important proenzymes include pepsinogen, trypsinogen and
chymotrypsinogen, which give rise to the proteolytic digestive enzymes.

B. Allosteric regulation
Includes: allosteric regulation, regulation by reversible covalent modification and
regulation by control proteins

Reversible covalent modification is a major mechanism for the rapid and transient
regulation of enzyme activity. Example: E. phosphorylation

- These are enzymes that change their conformational (shape) ensemble upon
binding of an effector (activator or inhibitor), which results in an apparent
change in binding affinity at a different ligand-binding site.

- This "action at a distance" through binding of one ligand affecting the binding
of another at a distinctly different site, is the essence of the allosteric
concept.

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 - Most (but not all) allosteric enzymes are oligomeric (consisting of multiple
subunits); the effectors that modulate the activity of these allosteric enzymes
are of two types (inhibitors or activators).
Most of them are oligomeric because quaternary structure provide more
flexibility.

- There are two ways that enzymatic activity can be altered by effectors: the
Vmax can be increased or decreased, or the Km can be raised or lowered.

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 - The ligand for hemoglobin is O2.
- kM for myoglobin is lower than kM for hemoglobin.
- All the four subunits are connected with each other; the activation of one
activates another. Therefore, hemoglobin subunits binds to O2 one by one.
- Allosteric Modulation (Cooperativity): Cooperativity is a phenomenon
displayed by enzymes or receptors that have multiple binding sites where
the affinity of the binding sites for a ligand is increased, positive
cooperativity, or decreased, negative cooperativity, upon the binding of a
ligand to a binding site.

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 Chapter 5
Carbohydrates
(Sugars)

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  Introduction
- Carbohydrates are carbon compounds that contain large quantities of hydroxyl
groups (-OH groups).
- The simplest carbohydrates also contain either an aldehyde group or a ketone
group.

(Aldose sugars) (Ketose sugars)

- The presence of the hydroxyl groups allows carbohydrates to interact with the
aqueous environment and to build hydrogen bonding, both within and between
chains.
 Derivatives of the carbohydrates can contain nitrogens, phosphates and
sulfur compounds.
 Carbohydrates also can combine with lipid to form glycolipids or with
protein to form glycoproteins.

- Carbohydrates can be classified as either:

1. Monosaccharides (monomers of carbohydrates)
2. Disaccharides
3. Oligosaccharides: from two to ten monosaccharide units, linked by glycosidic
bonds.
Disaccharides are considered oligosaccharides. Hence, every disaccharide is
oligosaccharide, but not every oligosaccharides is disaccharide.
4. Polysaccharides: hundreds of monosaccharide units, linked by glycosidic
bonds.

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  Carbohydrate nomenclature
- The predominant carbohydrates encountered in the body are structurally related to
the aldotriose glyceraldehyde and to the ketotriose dihydroxyacetone.
- All carbohydrates contain at least one asymmetrical (chiral) carbon and are,
therefore, optically active. With a few exceptions, those carbohydrates that are of
physiological significance exist in the D-conformation.
- The mirror-image conformations, called enantiomers, are in the L-conformation.

- D-Glyceraldehyde is an aldotriose and is it the simplest aldose.

- We start numbering from the closest point to the double bond.
- The number of the chiral carbon n-1.

 Monosaccharides
- The monosaccharides commonly found in humans are classified according to the
number of carbons they contain in their backbone structures. The major
monosaccharides contain four to six carbon atoms.
- Monosaccharides are either ketose or aldose.
- Monosaccharides chemical formula (CH2O)n

(Aldose sugars) (Ketose sugars)

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 - Aldoses

The number of
carbonyl
group in
aldoses is 1

- D-aldoses

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 - Ketoses

Simplest Ketose
The number of
carbonyl group
in ketoses is 2

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 - The aldehyde and ketone moieties of the carbohydrates with five and six carbons
will spontaneously react with alcohol groups present in neighboring carbons to
produce five- or six rings.

Anomeric OH

Anomeric carbon

 Disaccharides
- Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the hydroxyl of
a second sugar (or another alcohol-containing compound) are termed glycosidic
bonds, and the resultant molecules are glycosides.
- The linkage of two monosaccharides to form disaccharides involves a glycosidic
bond. Several physiogically important disaccharides are sucrose, lactose and
maltose.
- Glycosidic linkage is between anomeric hydroxyl and another hydroxyl group.

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 1. Sucrose: is composed of glucose and fructose through an α-(1,2)β-glycosidic
bond.

2. Lactose: is found exclusively in the milk of mammals and consists of galactose

and glucose in a β-(1,4) glycosidic bond.

3. Maltose: the major degradation product of starch, is composed of 2 glucose

monomers in an α-(1,4) glycosidic bond.

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  Polysaccharides
- Most of the carbohydrates found in nature occur in the form of high molecular
weight polymers called polysaccharides.
- The monomeric building blocks used to generate polysaccharides can be varied; in
all cases, however, the predominant monosaccharide found in polysaccharides is
D-glucose.
- When polysaccharides are composed of a single monosaccharide building block,
they are termed homopolysaccharides. Polysaccharides composed of more than
one type of monosaccharide are termed heteropolysaccharides.

1. Glycogen
- Glycogen is the major form of stored carbohydrate in animals.
- This crucial molecule is a homopolymer of glucose in α-(1,4) linkage; it is also
highly branched, with α-(1,6) branch linkages occurring every 8-10 residues.
(glycogen is always branched)
- Glycogen is a very compact structure that results from the coiling of the
polymer chains. This compactness allows large amounts of carbon energy to
be stored in a small volume, with little effect on cellular osmolarity.

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 2. Starch
- Starch is the major form of stored carbohydrate in plant cells.
- Its structure is identical to glycogen, except for a much lower degree of
branching (about every 20-30 residues).
- Amylose: Unbranched starch.
- Amylopectin: Branched starch.

3. Cellulose
- Cellulose is an example of structural polysaccharides.
- Cellulose is the most abundant organic compound on earth.
- It is made of glucose, like starch, but they differ in the type of 1-4 linkage.
- Instead of an α linkage as in starch, cellulose contains a β 1-4 linkage
- Straight and had trans CH2OH.

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122 | P a g e
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 Chapter 6
Metabolism

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  General introduction
- Living organisms require a continual input of free energy to maintain a state out of
equilibrium with the environment (2nd law of thermodynamics).

- Phototrophs: Use energy from the sun to convert energy-poor molecules into
energy-rich molecules.
- Chemotrophs: Obtain energy by oxidizing the energy-rich molecules made by the
phototrophs.

- 1st law of thermodynamics

Energy from photosynthesis or the oxidation of fuels can be transformed into:
 Mechanical work-muscle contraction
 Power biosynthesis
 An unequal distribution of ions across a biological membrane (chemical
potential) which could be used to:
1. Oxidative phosphorylation to make ATP (H+ gradients).
2. Active transport across membranes (transporters).
3. Nerve transmission (Na+/ K+ gradients).

- Metabolism includes all chemical reactions of cells and tissues.

- Metabolism consists of two types of reactions:
1. Synthesis: Anabolism
2. Degradation: Catabolism

A B

Catabolism and anabolism are usually not allocated together.

‫ال يمكن حصولها معا‬

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125 | P a g e
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  Metabolism has the following rules:
1. Build of organic molecules.
2. Water is the universal solvent.
3. Phosphate is the carrier of chemical energy.
4. Enzymes are the catalytic power.
5. Nucleic acids are the carrier of genetic information.
6. Most reactions are reversible -> ΔG is zero for a reversible reaction (except energy
producing reactions -> ΔG is negative).

7. ATP is the energy carrier: ATP <-> ADP +Pi, ∆G = -7.3Kcal/mol.

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 8. NADH and NADPH are the main electron carrier of the metabolism:
 Food oxidation -> CO2 + electrons
 Electrons + O2 -> H2O + ATP
 Electrons are not free and are transferred via special carrier
NAD+ + two electrons + H+ -> NADH
 NADH produces via the mitochondrial oxidative phosphorylation 3 ATP

 FADH2 produces via the mitochondrial oxidative phosphorylation 2 ATP

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 9. All molecules undergo a turnover. For example:
 Liver
Proteins have a half time of 5-6 days
Glycogen has a half time of 0.5-1 days
Phospholipids have a half time of 1-2 days

 Muscles
Proteins have a half time of 30 days
Glycogen has a half time of 0.5-1 days

 Brain
Phospholipids have a half time of 200 days

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  Metabolism of Carbohydrates
- Dietary carbohydrates from which humans gain energy enter the body in complex
forms, such as disaccharides and the polymers starch (amylose and amylopectin) and
glycogen. The polymer cellulose is also consumed but not digested.

- The first step in the metabolism of digestible carbohydrate is the conversion of the
higher polymers to simpler, soluble forms that can be transported across the
intestinal wall and delivered to the tissues.

Polymers (disaccharides, starch, glycogen)  monomers (monosaccharides)

- The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic
pH of 6.8 and contains lingual amylase that begins the digestion of carbohydrates.
The action of lingual amylase is limited to the area of the mouth and the esophagus;
it is virtually inactivated by the much stronger acid pH of the stomach.

- In the stomach, acid hydrolysis contributes to its degradation; the mixture of gastric
secretions, saliva, and food moves to the small intestine.

- The main polymeric-carbohydrate digesting enzyme of the small intestine is α-

amylase. This enzyme is secreted by the pancreas and has the same activity as
salivary amylase, producing disaccharides and trisaccharides.

- Monosaccharides are produced by intestinal saccharidases: sucrase, lactase, and

trehalase (Trehalose= alpha-linked disaccharide formed by an α,α-1,1-glucoside
bond between two α-glucose units).

- The net result is the almost complete conversion of digestible carbohydrate to its
constituent monosaccharides (glucose and other monosaccharide).

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 - Monosaccharides are transported across the intestinal wall to the hepatic portal
vein and then to liver. There they are converted to fatty acids, amino acids, and
glycogen, or oxidized by the various catabolic pathways of cells

 Glycolysis
- Partial oxidation of glucose is known as glycolysis. Glucose is oxidized to either
lactate or pyruvate.
- Under aerobic conditions, the dominant product in most tissues is pyruvate and the
pathway is known as aerobic glycolysis.
- During prolonged vigorous exercise, the dominant glycolytic product in many tissues
is lactate and the process is known as anaerobic glycolysis

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 - Anaerobic metabolism is faster than aerobic metabolism.
- Aerobic is more efficient.
- Anaerobic respiration is also called fermintation.
- Two types of fermintation:
1. Ethanol fermintation: in the bacteria.
E. coli in our body can perform ethanol fermintation.
2. Lactate fermintation: in the muscles, and RBCs.
Erythrocytes perform anerboic respiration because they lack mitochondria.

- Why is glucose the common fuel molecule?

Glucose is recognized by everything in the body (universal language).
(Virtually all cells are able to take up and utilize glucose)
- Why generating ATP through glycolysis and not through direct oxidation?
Gives more ATP with less time.
- Why is the inefficient glycolysis process so abundant?
Because it is faster.

- Glycolysis is a sequence of reactions that converts glucose into pyruvate.

 Production of small amount of ATP
 All intermediates are either C3 or C6
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 - The Energy Derived from Glucose Oxidation
The conversion of one mole of glucose to two moles of pyruvate is accompanied by
the net production of two moles each of ATP and NADH.

Glucose + 2 ADP + 2 NAD+ + 2 Pi -> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via
oxidative phosphorylation, producing three equivalents of ATP.
The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is 8
moles of ATP.
Complete oxidation of the 2 moles of pyruvate, through the KREBS cycle, yields an
additional 30 moles of ATP; the total yield, therefore being 38 moles of ATP from
the complete oxidation of 1 mole of glucose to CO2 and H2O.

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  Three stages in glycolysis
- Glycolysis has 10 steps.
- 7 of them are reversible.
- 3 of them are irreversible
(1.3.10)

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1. Energy investment
The chemical priming phase requiring 2ATP:
Take 2 ATP to produce 4 ATP.
Glucose  fructose-1,6-bisphosphate (F-1,6-BP).

 The first reaction of the first stage

The ATP-dependent phosphorylation of glucose to form glucose-6-phosphate (G6P) is
the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as
hexokinases.

- Hexokinase transfer phosphate group from ATP to the carbon.

- Mg2+ is a cofactor, and help with the transfer of phosphate group.

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 - The phosphorylation accomplishes two goals:
1. The hexokinase reaction converts nonionic glucose into an anion (has a 2-
charge) that is trapped in the cell, since cells lack transport systems for
phosphorylated sugars.
2. Glucose becomes activated to be capable of being further metabolized. (can
react more)
3. Drives more glucose uptake (outside there is glucose, inside no glucose -> leads
to more glucose coming inside the cell (concentration gradient)).

- The phosphate group is added to the outside carbon (carbon #6).

- Glucose is in the blood.

- Glucose-6-Phosphate is inside the cell.

- The first step -> ∆G > 0

The conversion of ATP to ADP gave the reaction 7.3Kcal/mol.
The reaction needed less than 7.3Kcal/mol, that’s why ∆G > 0

- Induced fit-mechanistic implications (mechanism of action)

Catalysis by proximity: Upon closure, the C6 hydroxyl (hydroxyl group of the sixth
carbon) of the bound glucose is brought close to the terminal phosphate of ATP.

Preventing undesired reactions: water is excluded from the active site. This prevents
the enzyme from catalyzing ATP hydrolysis.

Destabilization effect: the environment around the bound glucose becomes more
non-polar which favors the donation of the γ Pi of ATP.
Other Kinases use the same technique

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 - Hexokinase are Glucokinase isozymes
Glucokinase (hexokinase IV) is found in hepatocytes.
Hexokinase is found in nonhepatic tissues e.g. muscles (hexokinase I-III)

The Km: Physiological blood glucose ->

Hexokinase is 0.1mM 80-100 mg/dL
Glucokinase is 10mM (liver is slower)
4.5-5.5 mmol/L (mM)

- This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly
and efficiently trap blood glucose within their cells by converting it to glucose-6-
phosphate.

- One major function of the liver is to deliver glucose to the blood and this in ensured
by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is
sufficiently higher than the normal circulating concentration of glucose (4.5-5.5mM).

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 - This feature of hepatic glucokinase allows the liver to buffer blood glucose:
1. After meals (blood glucose levels are high) -> liver glucokinase is significantly
active  liver traps and stores circulating glucose.
2. When blood glucose falls to very low levels:
Tissues such as the brain, which are critically dependent on glucose, continue
to use blood glucose using their low Km hexokinases.

At high blood glucose levels, G6P inhibits (feedback inhibition -> products inhibits its
own synthesis) hexokinase I-III (present in muscle..etc).
Which prevents these tissues from taking glucose at high levels.

Liver Other tissues

Low glucose X ✓
High glucose ✓ X

- Under various conditions of glucose deficiency, such as long periods between meals,
the liver is stimulated to supply the blood with glucose through the pathway of
gluconeogenesis.
The levels of glucose produced during gluconeogenesis are insufficient to activate
glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.

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  The second reaction of the first stage:
Glucose doesn’t always have the ability to bind to two phosphate groups, that’s why it
is converted into fructose which has the ability to bind to two phosphate groups.

The second reaction of glycolysis is an isomerization (structural isomerization):

G6P (aldose)  fructose-6-phosphate (F6P) (ketose)
This reaction is freely reversible.

Notice F6P has two outside carbons that allow it to bind to two phosphate groups,
which will increase its energy.

1 2 3

The mechanism involves ring opening, isomerization and ring closure.

1 2 3
The enzyme catalyzing this reaction is phosphohexose isomerase (also known as
phosphoglucose isomerase).

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  The third reaction of the first stage:
Attaching another phosphate to F-6-P (another ATP molecule is consumed).

F6P + ATP  fructose-1,6-bisphosphate (F-1,6-BP) + ADP

- F-6-P has a high energy and waiting for a reaction to happen.

- This reaction is catalyzed by phosphofructokinase-1 or PFK-1.

This reaction is irreversible.

- PFK has a mechanism similar to that of Hexokinase.

- The Phosphofructokinase reaction determines the pace of Glycolysis, because unlike
G-6-P reaction, it is committed to glycolysis and doesn’t undergo other reactions.
- The enzyme is highly-regulated.

Bisphosphate = 2 separate Pi groups

Diphosphate = 2 attached Pi groups (with anhydride bond)

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2. Hexose – triose splitting
Second stage of Glycolysis: 6-carbon sugar is cleaved into two 3-carbon fragments.

 The first reaction of the second stage:

Aldolase catalyses the hydrolysis of F-1,6-BP into two 3-carbon products:

F-1,6-BP  dihydroxyacetone phosphate (DHAP) + glyceraldehyde-3- phosphate

(GAP).

Ketose

Aldose

This reaction occurs in linear/chain form.

The aldolase reaction is reversible

Glycolysis pathway continues from GAP and not from DHAP!

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  The second reaction of the second stage:
The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by
triose phosphate isomerase.

Triose phosphate IsoMerase (TIM) catalyzes:

Dihydroxyacetone-P  glyceraldehyde-3-P

- GAP proceeds in the glycolysis pathway. However, the isomerization reaction favors
dihydroxyacetone-P.
How then can we achieve throughput in the glycolysis pathway?
Since GAP is consumed, DHAP will be converted to GAP.

- Triose phosphate IsoMerase (TIM) structure

TIM structure is an αβ barrel.
Tertiary structure
His -> Histidine
Histidine is a proton donor
Glu -> Glutamate
Glutamate is a proton acceptor

Omega loop

In an αβ barrel there are 8 parallel β-strands surrounded by 8 α-helices.

Short loop connect alternating β-strands & α-helices

The structure reveals:

1. Substrate-binding site.
2. Important residues for catalysis.
3. An important loop (closes off the active site on S binding).
4. Other glycolytic enzymes, like aldolases and pyruvate kinase are TIM barrels.

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 - TIM mechanism

The ketose/aldose conversion involves acid/base catalysis. proceeds via an enediol

intermediate

Active site Glu and His residues are thought to extract and donate protons during
catalysis.

Histidine is a proton donor

Glutamate is a proton acceptor

Histidine gives the proton to the substrate, and then the proton goes to the
glutamate.
The proton then goes back to histidine in the same mechanism.

Histidine  substrate  glutamate.

The purpose of giving the proton is to apply some changes to the substrate
(ketose/aldose conversion)

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3. Energy and redox potential production

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  The first reaction of the third stage (redox potential) -> indirect ATP production:
The third phase of glucose catabolism features the energy-yielding glycolytic reactions
that produce ATP and NADH. In the first of these reactions is the oxidation of G3P to
1,3-bisphosphoglycerate (1,3-BPG):

Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:

Glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+

This overall reaction is the sum of two coupled reactions (energy coupling):
1. Exergonic oxidation of the aldehyde in GAP to a carboxylic acid.
2. The formation of an acyl phosphate (a "high energy" bond (~P)).

In this reaction, ATP is not necessarily produced:

Anaerobic  NADH stays in cytosol  Not further ATP production.
Aerobic  NADH goes to the mitochondria  ATP production (Oxidative
phosphorylation).

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  The second reaction of the third stage (direct ATP production):
The high-energy phosphate of 1,3-BPG is used to form ATP:

1,3-BPG +ADP  3-phosphoglycerate (3PG) + ATP

We used a phosphate group from 1,3-BPG to convert ADP to ATP (direct ATP
production)

This reaction is catalysed by phosphoglycerate kinase.

This is the only reaction of glycolysis that involves ATP and yet is reversible under
normal cell conditions.

This phosphate transfer reaction has low DG, since one ~P bond is cleaved & another
synthesized.
The enzyme undergoes substrate-induced conformational change similar to that of
Hexokinase.

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  The last reactions in the third stage (direct ATP production):
The remaining reactions of glycolysis are aimed at converting the relatively low
energy phosphoacyl-ester of 3-PG to a high-energy form and harvesting the
phosphate as ATP.

- Phosphoglycerate mutase catalyzes:

3-phosphoglycerate  2-phosphoglycerate
In general, mutases catalyzed intermolecular shift of chemical group (The enzyme
that does intra-molecular transfer is called mutase)
The transfer of phosphate group from carbon #3 to carbon #2 make it more active.

- Enolase catalyzes:
2-phosphoglycerate  phosphoenolypyruvate + H2O
The enol in PEP has higher transfer potential of the phosphoryl group. Therefore
allowing ATP formation in the next step.
Intermediate PEP  highly unstable
Will donate phosphate group to ADP, which will produce pyruvate.

- Pyruvate kinase catalyzes:

Phosphoenolpyruvate + ADP  pyruvate + ATP

Mutase-(His)-phospate + 3-phosphoglycerate  Mutase-(His) + 2,3bisphosphoglycerate

Mutase-(His) –phosphate + 2-phosphoglycerate

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  The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme
pyruvate kinase (PK).
This reaction is highly irreversible.

phosphoenoylpyruvate (PEP) + ADP  Pyruvate + ATP

Energy Yield of Glycolysis:

Glucose + 2Pi + 2ADP + 2NAD  2Pyruvate + 2ATP+ 2NADH + 2H2O + 2H+
2NADH = 2 x 3ATP = 6 ATP
Total = 8 ATP

How many ATP ~P bonds expended? 2 ATP molecules.

How many ~P bonds of ATP produced? 4 ATP molecules.
Net production of ~P bonds of ATP per glucose: 2 ATP molecules.

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  Anaerobic Glycolysis
- Under anaerobic conditions and in erythrocytes/muscles under aerobic conditions,
pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH).

Pyruvate + NADH  lactate + NAD+

Aerobic  pyruvate goes into the mitochondrion.

Pyruvate loses the CO2 and converts to acetyl. CoA binds to acetyl.
RBCs can’t perform this step because it lack mitochondria.

Bacterial fermentation

Anaerobic

Human fermentation

Anaerobic

The rate of ATP production from glycolysis is approximately 100X faster than from oxidative
phosphorylation.

Note: the amount of NAD+ and NADH in the cell is limited.

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 - In aerobic organisms:
Pyruvate produced in Glycolysis is oxidized to CO2 via Krebs cycle.
NADH produced in Glycolysis & Krebs Cycle is reoxidized via the respiratory chain,
with production of much additional ATP.

- Anaerobic organisms lack a respiratory chain.

They must reoxidize NADH produced in Glycolysis through other reactions: Alcohol
fermentation (e.g., yeast) Lactic acid fermentation
- The conversion of pyruvate to lactate provides the cell with a mechanism for the
oxidation of NADH (produced during the GAPDH reaction) to NAD+.

- This reduction is required since NAD+ is a necessary substrate for GAPDH

(glyceraldehyde-3-phosphate dehydrogenase), without which glycolysis will cease.

NAD+ achieved from vitamin niacin (B3),

with limited sources from diet, e.g.,
tuna, chicken, turkey, mushroom, etc

- Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred
to mitochondrial carriers of the oxidative phosphorylation pathway generating a
continuous pool of cytoplasmic NAD+.

- Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized
than anaerobic glycolysis.

- During exertion, muscle cells do not need to energize anabolic reaction pathways. The
requirement is to generate the maximum amount of ATP, for muscle contraction, in
the shortest period. This is why muscle cells derive almost all of the ATP consumed
during exertion from anaerobic glycolysis.

- The lactate produced during anaerobic glycolysis diffuses from the tissues and is
transported to highly aerobic tissues such as cardiac muscle and liver. The lactate is
then oxidized to pyruvate in these cells by LDH (lactate dehydrogenase) and the
pyruvate is further oxidized in the Krebs cycle.
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 - NAD+-binding region in dehydrogenases
Notice that the nicotinamide-binding half (yellow) is structurally similar to the
adenine-binding half (red).
The two halves together form a structural motif called a Rossmann fold.
The NAD+ molecules binds in an extended conformation.

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 - Glucose is the universal language between all cells of the body.
- Galactose and Fructose need specific tissues.

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 Chapter 7
Gluconeogenesis and control of glycolysis

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  Glucose is either:
1- Broken down  glycolysis  all tissues are capable of glycolysis.
2- Collected  Gluconeogenesis  not all tissues are capable of glycolysis.

Between these two processes, we have 7 mutual steps, which are the reversible
steps. In addition, we have 3 irreversible steps.

 Gluconeogenesis
- Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from
glycogen).

- Gluconeogenesis is a ubiquitous process, present in plants, animals, fungi, bacteria,

and other microorganisms.

- In animals, gluconeogenesis takes place mainly in the liver and, to a lesser extent, in
the cortex of kidneys.

- The production of glucose from other metabolites is necessary for use as a fuel
source by the brain, testes, erythrocytes and kidney since glucose is the sole energy
source for these organs.

- During starvation, however, the brain can derive energy from ketone bodies, which
are converted to acetyl-CoA.

- Synthesis of glucose from three and four carbon precursors is essentially a reversal
of glycolysis.

- The irreversible steps mediate the control of the gluconeogenesis.

- Gluconeogenesis has eleven steps.

Step 1  occurs in the mitochondria
Step 2-10  occurs in the cytosol.
Step 11  occurs in the ER (endoplasmic reticulum).

The reverse reaction of kinase is phosphatase

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  Galactosemia
Galactosemia is a disorder that affects how the body processes a simple sugar called
galactose.
Lactase
Lactose ----------------> Glucose + Galactose

When the galactose levels are high in the baby, and it cannot enter glycolysis, that is
when galactosemia occurs.

Normal process of Glucose

entering glycolysis

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  Two unique enzymes are involved in Fructose metabolism only in the liver.
1. Fructokinase
2. Fructose-1-phosphate aldolase

DHAP

GAP

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  Regulation of Glycolysis
- Tightly regulated for reasons of energy and metabolites supply.

- Hexokinase (step 1), Phosphofructokinase (step 3) & Pyruvate Kinase (step 10).

- Good candidates for regulation sites.

- Control of these enzymes determines the rate of the Glycolysis pathway.

- 1. Local control involves allosteric control of key pathway enzymes by metabolites

that signal the energy charge and building block status of the cell. (‫)داخل الخلية‬
The energy charge is related to ATP and AMP concentrations.  ATP/AMP
If ATP is high  We have high energy.
If AMP is high  We have low energy.
We start Glycolysis when the ATP is low.
We start Gluconeogenesis when the ATP is high.

We didn’t consider ADP because it represents a medium state that can’t be used as
a measure.

- 2a. Global covalent control involves signal transduction pathways that change
downstream enzymes’ activity (usually by phosphorylation). (‫)خارج الخلية‬
Hormonal signal  some stay the cytosol and the other enter the nucleus  signal
transduction pathway.

- 2b. Global transcriptional control involves signal transduction pathways that change
gene expression patterns. (‫)خارج الخلية‬
The hormonal signals can act in two ways:
1. Enhance gene expression of enzyme  activate the enzymes.
2. Inhibit gene expression of enzyme  de-activate the enzymes.

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 Cells
with
high
energy

Gluconeogenesis

- Glycolysis  Produce energy in the state of NO energy.

- Gluconeogenesis  Production of glucose in the state of energy.

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  Regulation of Glycolysis
Glycolysis is inhibited when the blood-glucose levels are low.
1. Regulation of hexokinase (HK) in muscle
Glycolysis’ first reaction is catalyzed by Hexokinase:
Glucose + ATP  glucose-6-P + ADP G = - 8.0 kcal mol-1

Hexokinase is inhibited by its product glucose-6phosphate.

Glucose-6-phosphate inhibits hexokinase by competition at the active site, as well as

by allosteric interactions at a separate site on the enzyme (local control).

What is the physiological role of this product inhibition?

Because the amount of energy needed is produced. If the glycolysis continuous it a
waste of energy and molecules.

HK isoform in the liver (GK), is not inhibited by its product. Its Km is 50 times higher
(low affinity) compared to the muscle or brain isoform.

2. Regulation of pyruvate kinase

Pyruvate Kinase catalyzes the last glycolytic reaction: controls the outflow from the
pathway.

Phosphoenolpyruvate (PEP) + ADP+H+  pyruvate + ATP DG= - 4.0 kcal*mole

Control by:
1) Allosteric regulation (ATP, Alanine and Fructose 1,6, Bisphosphate).
Accumulation of ATP and Alanine (products)  inhibition
Accumulation of Fructose 1,6 Bisphosphate (reactants)  activation

Local control is present in both the muscle and liver.

2) Covalent modification (phosphorylation).

Global control is only in the liver.

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 Global control

Local control

- Several isozymic forms of pyruvate kinase (a tetramer of 57-kd subunits) encoded

by different genes are present in mammals: the L type predominates in the liver,
and the M type in muscle and the brain.
The L and M forms of pyruvate kinase have many properties in common. Indeed,
the liver enzyme with regard to allosteric regulation. However, the isozymic forms
differ in their susceptibility to covalent modification. The catalytic properties of the
L-form, but not of the M forms, are also controlled by reversible phosphorylation.

- When the blood-glucose level is low, the glucagon-triggered cyclic AMP cascade
leads to phosphorylation of pyruvate kinase by activating protein kinase A, which
diminished its activity.
This hormone-triggered phosphorylation prevents the liver from consuming glucose
when it is more urgently needed by the brain and muscle.

- In additions, adrenaline (epinephrine) works on raising the blood-glucose levels in

the muscles.
Most of the time, adrenaline and glucagon causes phosphorylation of enzymes.

- When the blood-glucose levels are high, the insulin hormone works on lowering it.
Most of the time, insulin causes dephosphorylation of enzymes.
Dephosphorylation of enzymes actually taken place by Protein Phosphatase 1 (PP1)
Phosphorylation of enzyme actually taken place by Protein Kinase A (PKA)
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 3. Regulation of phosphofructokinase
Phosphofructokinase is the pace maker enzyme in the liver and muscles.

Phosphofructokinase (PFK) catalyzes the third reaction of glycolysis:

Fructose-6-P + ATP  fructose-1,6-bisP + ADP
G = - 5.3 kcal*mole-1
PFK in the liver is regulated similarly as in the muscle except for the differences
indicated.

Has four catalytic sites and four allosteric sites.

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 - A decrease in pH (H+) also inhibits phosphofructokinase activity by augmenting the
inhibitory effect of ATP. The pH might fall when muscle is functioning anaerobically,
producing excessive quantities of lactic acid.
The inhibitory effect protects the muscle from damage that would result from
accumulation of too much acid.

- Why is AMP and not ADP the positive regulator of phosphofructokinase?

When ATP is being utilized rapidly, the enzyme adenylate kinase can form ATP from
ADP by the following reaction:

Thus, some ATP is salvaged from ADP, and AMP becomes the signal for the low-
energy state. Moreover, the use of AMP as an allosteric regulator provides an
especially sensitive control. We can understand why by considering
1) That the total adenylate pool ([ATP], [ADP], [AMP]) in a cell is constant over the
short term.
2) That the concentration of ATP is greater than that of ADP and the
concentration of ADP is, in turn, greater than that of AMP.

Consequently, small-percentage changes in [ATP] result in large-percentage changes

in the concentrations of the other adenylate nucleotides. This magnification of small
changes in [ATP] to larger changes in [AMP] leads to tighter control by increasing
the range of sensitivity of phosphofructokinase.

- Why is phosphofructokinase the glycolysis pace maker enzyme?

When phosphofructokinase is inactive, the concentration of fructose-6-phosphatase
rises. In turn, the level of glucose-6-phosphate rises because it is in equilibrium with
fructose-6-phosphate. Hence, the equilibrium of phosphofructokinase leads to the
inhibition of hexokinase.

- Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis?

The reason becomes evident on noting that glucose-6-phosphate in not solely a
glycolytic intermediate. In muscles, glucose-6-phosphate can also be converted into
glycogen.
The first irreversible reaction is unique to the glycolytic pathway (the committed
step).

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 - Regulation with respect to ATP is the same in liver as muscles.

- Low pH (H+) is not a metabolic signal for the liver enzyme, because lactate is not
normally produced in the liver. Indeed, as we will see, lactate is converted into
glucose in the liver.

- Glycolysis also furnishes carbon skeletons for biosyntheses, and so a signal

indicating whether building blocks are abundant or scarce should regulate
phosphofructokinase.

- In the liver, phosphofructokinase is inhibited by citrate, an early intermediate in the

citric acid cycle. A high level of citrate in the cytoplasm means that biosynthetic
precursors are abundant, and so there is no need to degrade additional glucose for
this purpose. Citrate inhibits phosphofructokinase by enhancing the inhibitory
effect of ATP.

- One means by which glycolysis in the liver responds to changes in blood glucose is
through the signal molecules fructose 2, 6-bisphosphate, a potent activator of
phosphofructokinase. In the liver, the concentration of fructose-6-phosphate rises
when blood-glucose concentration is high, and the abundance of fructose-6-
phosphate accelerates the synthesis of fructose 2,6 bisphosphate.
Hence, the abundance of fructose-6-phosphate leads to a higher concentration of
fructose 2,6 bisphosphate.

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 - Glucose transporter (GLUT) is a facilitative transport protein involved in glucose
translocation across the cell membrane.

- Several glucose transporters mediate the thermodynamically downhill movement

of glucose across the plasma membranes of animal cells.

- Each glucose transporter has a 12-transmembrane-α-helix structure.

- The binding site is an omega loop.

- On the surface of asparagine occurs the binding of glucose.

- When the glucose enters pancreatic β cells, it mediates the exocytosis of insulin.

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 The members of this family have distinctive roles (‫)شرح الكتاب‬
1) GLUT1 and GLUT3, present in nearly all mammalian cells, are responsible for
basal glucose uptake. Their KM value for glucose is about 1 mM, significantly less
than the normal serum-glucose level, which typically ranges from 4 mM to 8 mM.
Hence, GLUT1 and GLUT3 continually transport glucose into cells at an essentially
constant rate.

2) GLUT2, present in liver and pancreatic β cells, is distinctive in having a very high
KM value for glucose (15–20 mM). Hence, glucose enters these tissues at a
biologically significant rate only when there is much glucose in the blood. The
pancreas can sense the glucose level and accordingly adjust the rate of insulin
secretion. The high KM value of GLUT2 also ensures that glucose rapidly enters
liver cells only in times of plenty.

3) GLUT4, which has a KM value of 5 mM, transports glucose into muscle and fat
cells. The number of GLUT4 transporters in the plasma membrane increases
rapidly in the presence of insulin, which signals the fed state. Hence, insulin
promotes the uptake of glucose by muscle and fat. Endurance exercise training
increases the amount of this transporter present in muscle membranes.

4) GLUT5, present in the small intestine, functions primarily as a fructose

transporter.

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  Gluconeogenesis: Synthesis of glucose from pyruvate.
- Gluconeogenesis occurs mainly in liver and cortex of the kidney.

- Non-carbohydrate precursors are first converted into pyruvate or enter the

pathway at a later stage: major precursors: lactate, amino acids, and glycerol.

- Gluconeogenesis is not the reverse of glycolysis.

- Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis.

- Three Glycolysis reactions have such a large negative ∆G that they are essentially
irreversible:
1) Hexokinase/glucokinase
2) Phosphofructokinase -1 (PFK-1)
3) Pyruvate Kinase
These steps must be bypassed in Gluconeogenesis.

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  Bypass 1: Pyruvate to Phosphoenolpyruvate
- In Glycolysis:
Pyruvate Kinase catalyzes:
Phosphoenolpyruvate (PEP) + ADP  pyruvate + ATP ∆G = -7.5

- In gluconeogenesis:
Conversion of pyruvate to PEP requires the action of two mitochondrial enzymes.
1) Pyruvate carboxylase (PC). As the name of the enzyme implies, pyruvate is
carboxylated to form oxaloacetate (OAA). The CO2 in this reaction is in the form
of bicarbonate (HCO3-).

Pyruvate + CO2 + H2O + ATP  oxaloacetate + ADP + Pi + 2H+

2) PEP carboxykinase (PEPCK): this enzyme catalyzes the conversion of

oxaloacetate to phosphoenolpyruvate (PEP).

Oxaloacetate + GTP  phosphoenolpyruvate (PEP) + GDP + CO2

- PEPCK requires GTP in the decarboxylation of OAA to yield PEP. Since PC

incorporated CO2 into pyruvate and it is subsequently released in the PEPCK
reaction, no net fixation of carbon occurs. Human cells contain almost equal
amounts of mitochondrial and cytosolic PEPCK so this second reaction can occur in
either cellular compartment.

- OAA used in the cytoplasm for gluconeogenesis is formed in the mitochondrial

matrix by carboxylation of pyruvate.
For gluconeogenesis to proceed, the OAA produced by PC needs to be transported
to the cytosol. However, no transport mechanism exist for its' direct transfer and
OAA will not freely diffuse. Mitochondrial OAA can become cytosolic via reduction
to malate, which is transported to the cytosol.

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 - Mitochondrial OAA can be reduced to malate in a reversal of the Krebs cycle
reaction catalyzed by malate dehydrogenase (MDH). The reduction of OAA to
malate requires NADH, which will be accumulating in the mitochondrion as the
energy charge increases.

- The resultant malate is transported to the cytosol where it is oxidized to OAA by

cytosolic MDH.

The net result of the PC and PEPCK reactions is:

Pyruvate + ATP + GTP + H2O  PEP + ADP + GDP + Pi + 2H+

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 PC is not expressed
in muscle

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 1. First reaction (in mitochondria) (irreversible step)
Pyruvate Carboxylase (PC) a two-step mechanism.
PC is composed of three domains: biotin, ATP and pyruvate binding domains.
Biotin  Vitamin B7

- First step: biotin carboxylation aided by ATP and CO2

Biotin-enzyme + HCO3- + ATP ⇔ CO2-biotin-enzyme + Pi + ADP

- Second step: carboxyl transfer to pyruvate

CO2-biotin-enzyme + pyruvate ⇔ biotin-enzyme + oxaloacetate (+ CO2) DG~-5kcal/mol
(Occurs spontaneously)

- Pyruvate carboxylase is a special interest because of its structural, catalytic, and

allosteric properties. The N-terminal 300 to 350 amino acids from an ATP-grasp
domain, which is an ATP-activating domain found in many enzymes, to be
considered in more detail when we examine nucleotide biosynthesis. The C-
terminal 80 amino acids constitute a biotin-binding domain that we will see again in
fatty acid synthesis.

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 2. Second (cytosolic) reaction of the first bypass (reversible step)

- Why carboxylation and decarboxylation is required to drive PEP formation?

Decarboxylation often drives reactions that are otherwise highly endergonic.
In short, decarboxylation aids with adding phosphate groups and make it easier and
less energy consumed.

- PEP Carboxykinase (PEPCK) catalyzes oxaloacetate conversion to PEP. It is thought

to proceed in two steps:

First  Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion

intermediate.

Second  Phosphate transfer from GTP then yields phosphoenolpyruvate (PEP).

Overall for the first bypass: G ~0

Pyruvate + ATP + GTP + H2O → phosphoenolpyruvate + ADP + GDP + Pi + 2H+

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  Bypass 2: Fructose-1,6-bisphosphate to Fructose-6-phosphate (occur in the cytosol)
- In Glycolysis:
Phosphofructokinase catalyzes:
Fructose-6-P + ATP  fructose-1,6-bisP + ADP ∆G= -5.3
Endergonic reaction.

- In Gluconeogenesis:
Fructose-1,6-bisphosphatase catalyzes:
Fructose-1,6-bisP + H2O  fructose-6-P + Pi
Exergonic reaction.

- Fructose-1,6-bisphosphate (F1,6BP) conversion to fructose-6-phosphate (F6P) is the

reverse of the rate limiting step of glycolysis. The reaction, a simple hydrolysis, is
catalyzed by fructose-1,6-bisphosphatase (F1,6BPase).
- Like the regulation of glycolysis occurring at the PFK-1 reaction, the F1,6BPase
reaction is a major point of control of gluconeogenesis.

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  Bypass 3: Glucose-6-phosphate (G6P) to Glucose (or Glycogen)
- In glycolysis
Hexokinase catalyzes:
Glucose + ATP  glucose-6-phosphate + ADP ∆G= -8

- In Gluconeogenesis:
Glucose-6-Phosphatase catalyzes:
Glucose-6-phosphate + H2O  glucose + P

This reaction takes place mainly in the liver and kidney. Glucose-6-Phosphatase is
expressed only there.
In other tissues, gluconeogenesis ends at G6P.

- G6P is converted to glucose through the action of glucose-6-phosphatase (G6Pase).

This reaction is also a simple hydrolysis reaction like that of F1,6BPase. Since the
brain and skeletal muscle, as well as most non-hepatic tissues, lack G6Pase activity,
any gluconeogenesis that occurs in these tissues is not utilized for blood glucose
supply.

- In the kidney, muscle and especially the liver, G6P can be shunted toward glycogen
if blood glucose levels are adequate.

- Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER)

membrane in liver cells.
The catalytic site is found to be exposed to the ER lumen. Other subunits may
function as a translocase, providing access of substrate to the active site.

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 - The last step of gluconeogenesis is the release of glucose.

Wanted**

- The generation of free glucose in an important control point.

The fructose-6-phosphate generated by fructose 1,6 bisphosphatase is readily
converted into glucose-6-phosphate. In most tissues, gluconeogenesis ends here.
Free glucose is not generated; rather, the glucose-6-phosphate is processed in some
other fashion, notably to form glycogen. One advantage to ending gluconeogenesis
at glucose-6-phosphate is that, unlike free glucose, the molecule is not transported
out of the cell. To keep glucose inside the cell, the generation of free glucose is
controlled in two ways.
1) First, the enzyme responsible for the conversion of glucose-6-phosphate into
glucose, which is glucose-6-phosphatase, is regulated.

2) Second, the enzyme is present only in tissues whose metabolic duty is to

maintain blood-glucose homeostasis- tissues that release glucose into the
blood. These tissues are the liver and to a lesser extent the kidney.

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 - Glycolysis & Gluconeogenesis are both spontaneous.

Glycolysis:
Glucose + 2NAD + 2ADP + 2 Pi  2pyruvate + 2NADH + 2ATP+ 2H20+ 2H+
DG = -20 Kcal/mol

- Gluconeogenesis:
2pyruvate + 2NADH + 4ATP + 2GTP + 6H20  glucose + 2NAD+ 4ADP + 2GDP + 6Pi + 2H+
DG =-9 Kcal/mol

- Questions:
1. Glycolysis yields how many ~P? 2
2. Gluconeogenesis expends how many ~P? 6
3. In both pathways, the cell wastes, how many ~P per cycle? 4

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 - The stoichiometry of gluconeogenesis

- In contrast, the stoichiometry for the reversal of glycolysis is:

- We need 3 glycolysis processes to perform one gluconeogenesis.

Explanation:
Glycolysis produces only two net molecules of ATP per 1 molecule of glucose.
Gluconeogenesis requires an input of six equivalents of ATP or GTP for each
molecule of glucose.

- Note that six nucleoside triphosphate molecules are hydrolyzed to synthesize

glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are
generated in glycolysis in the conversion of glucose into pyruvate. Thus, the extra
cost of gluconeogenesis is four high-phosphoryl-transfer-potential molecules for
each molecule of glucose synthesized from pyruvate. The four additional molecules
having high phosphoryl-transfer potential are needed to turn an energetically
unfavorable process (the reversal of glycolysis) into a favorable one
(gluconeogenesis). Here we have a clear example of the coupling of reactions: NTP
hydrolysis is used to power an energetically unfavorable reaction.

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Summary of gluconeogenesis reactions.

- To prevent this waste,

Glycolysis & Gluconeogenesis are reciprocally regulated.

- Two levels of regulation: local and global

1. Local Control: reciprocal allosteric regulation by adenine nucleotides (energy charge)

and intermediate metabolites.

- In Glycolysis:
Phosphofructokinase is inhibited by ATP and stimulated by AMP.

- In Gluconeogenesis:
Fructose 1,6-bisphosphatase is inhibited by AMP and stimulated by ATP.

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 2. Global Control (through GPCRs signaling cascade)
- Global control in liver cells includes reciprocal effects of a cyclic AMP cascade,
triggered by the hormone glucagon when blood glucose is low.
Phosphorylation of enzymes & regulatory proteins in the liver by Protein Kinase A
results in:
1) Inhibition of glycolysis
2) Stimulation of gluconeogenesis, making glucose available for release to the
blood (for the brain use e.g.)

- Enzymes relevant to these pathways that are phosphorylated by PKA include:

1) Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated.
2) CREB (cAMP response element binding protein) which activates transcription
of the gene for PEP Carboxykinase (gluconeogenesis increased).
3) A bi-functional enzyme that makes and degrades an extremely important
allosteric regulator: fructose-2,6-bisphosphate.

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  Reciprocal regulation by the allosteric modulator
Fructose-2,6-bisphosphate

- In glycolysis:
Fructose-2,6-bisphosphate allosterically activates the glycolytic enzyme
Phosphofructokinase.

- In gluconeogenesis:
Fructose-2,6-bisphosphate inhibits the gluconeogenesis enzyme Fructose-1,6-
bisphosphatase.

- A bi-functional enzyme makes and degrades the allosteric regulator, fructose-2,6-

bisphosphate depending on its phosphorylation state.
Dephosphorylation  high blood-glucose levels.
Phosphorylation  low blood-glucose levels.

Gluconeogenesis, a second source of glucose, is stimulated by glucagon via two

mechanisms:
1. Reduction of fructose-2,6-bisphosphatase (F2,6-BP) formation. Reduced F2,6-BP
synthesis simultaneously removes the stimulation of phosphofructokinase-1 while
increasing the activity of F1,6-BP. This results in an increase in conversion of F1,6-BP
to F6P.
2. Inactivation of pyruvate kinase. Phosphorylation of pyruvate kinase by protein
kinase A reduces futile recycling of phosphoenolpyruvate back to pyruvate. Instead
phosphoenolpyruvate is converted to F1,6-BP through reverse glycolysis. Pyruvate
kinase is further inhibited by alanine and adenosine triphosphate (ATP), both of
which are elevated during gluconeogenesis.
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  Cori Cycle

- The Cori cycle costs 6 Phosphates in liver for every 2 Phosphates made available in
muscle. The net cost is 4 Phosphates.
- Although costly in Phosphates bonds, the Cori Cycle allows the organism to
accommodate to large fluctuations in energy needs of skeletal muscle between rest
and exercise.

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  Substrates for Gluconeogenesis
- Lactate:
Lactate is the main source for glucose synthesis by gluconeogenesis. During
anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate
dehydrogenase (LDH).
This reaction serves two critical functions during anaerobic glycolysis.
1) In the direction of lactate formation the LDH reaction requires NADH and yields
NAD+ which is then available for use by the glyceraldehyde-3-phosphate
dehydrogenase reaction of glycolysis. These two reactions are, therefore,
intimately coupled during anaerobic glycolysis.

2) The lactate produced by the LDH reaction is released to the blood stream and
transported to the liver where it is converted to glucose. The glucose is then
returned to the blood for use by muscle as an energy source and to replenish
glycogen stores.

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 - Pyruvate:
Pyruvate, generated in muscle and other peripheral tissues, can be transaminated
to alanine which is returned to the liver for gluconeogenesis. Alanine is used here as
NH4 transporter to the liver where it is removed via the urea cycle.

- Amino Acids:
All 20 of the amino acids, excepting leucine and lysine, can be degraded to Krebs
cycle intermediates as discussed in the metabolism of amino acids.

This allows the carbon skeletons of the amino acids to be converted to oxaloacetate
and subsequently into pyruvate.

The pyruvate thus formed can be utilized by the gluconeogenic pathway.

When glycogen stores are depleted, in muscle during exertion and liver during
fasting, catabolism of muscle proteins to amino acids contributes the major source
of carbon for maintenance of blood glucose levels.

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  The hydrolysis of triacylglycerol in fat cells yields glycerol and fatty acids. Glycerol is a
precursor of glucose, but animals cannot convert fatty acids into glucose, for reasons
that will be given later. Glycerol may enter either the gluconeogenic or the glycolytic
pathway at dihydroxyacetone phosphate.

 The major site of gluconeogenesis is the liver, with a small amount also taking place
in the kidney. Little gluconeogenesis takes place in the brain, skeletal muscle, or heart
muscle. Rather, gluconeogenesis in the liver and kidney helps to maintain the glucose
level in the blood so that the brain and muscle can extract sufficient glucose from it
to meet their metabolic demands.

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 Chapter 8
The Citric Acid Cycle
&
Oxidative phosphorylation

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  The citric acid cycle (Krebs cycle/Tricarboxylic acid cycle)
- The fate of pyruvate, the end product of glycolysis, depends on the cell energy
needs.

1. In cells or tissues with a high energy, pyruvate is directed toward gluconeogenesis.

2. In cells or tissues with a low energy pyruvate is preferentially oxidized to CO2 and
H2O in the Tricarboxylic acid cycle (TCA) (also known as Krebs cycle or Citric acid
cycle):
1) Production of 15 ATP per pyruvate.
2) Takes place in the mitochondrion.

3. The conversion to pyruvate to acetyl-CoA (coenzyme A) is the linkage between

glycolysis and the Krebs cycle:

Pyruvate dehydrogenase
+
Pyruvate + CoA + NAD ------------------------------ CO2 + acetyl-CoA + NADH

The oxidative decarboxylation occurs in the mitochondrial matrix.

In humans, CoA biosynthesis requires cysteine, pantothenate, and adenosine

triphosphate (ATP).

The formation of acetyl CoA from carbohydrates is less direct than from fat.
The pyruvate carrier, (antiporter), transports pyruvate into mitochondria in
exchange for OH-.

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  The formation of Acetyl CoA from pyruvate

1. Decarboxylation
Pyruvate combines with TPP and is then decarboxylated to yield hydroxyethyl-TTP

This reaction is catalyzed by the pyruvate dehydrogenase component (E1) of the

multienzyme complex. TTP is the prosthetic group of the pyruvate dehydrogenase
component.

2. Oxidation
The hydroxyethyl group attached to TTP is oxidized to from an acetyl group while
being simultaneously transferred to lipoamide, a derivative of lipoic acid that is
linked to the side chain of a lysine residue by an amide linkage.

Note than this transfer results in the formation of an energy-rich thoiester bond.

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 3. Formation of Acetyl CoA
The acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA.

Dihydrolipoyl transacetylase (E2) catalyzes this reaction. The energy-rich thioester

bond is preserved as the acetyl group is transferred to CoA.
Recall that CoA serves as a carrier of many activated acyl groups, of which acetyl is
the simplest. Acetyl CoA, the fuel for the citric acid cycle, has now been generated
from pyruvate.

- The pyruvate dehydrogenase complex cannot complete another catalytic cycle until
the dihydrolipoamide is oxidized to lipoamide. In a fourth step, the oxidized form of
lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3). Two electrons are
transferred to a FAD prosthetic group of the enzyme and then to NAD+.

- These lipoamides serve as acceptors for the acetyl residues from pyruvate, transfer
them to acetyl-CoA, and reduce lipoamide to dihydrolipoamide in the process.

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 - The citric acid cycle removes electrons from acetyl CoA and uses these electrons to
form NADH and FADH2.

- Three hydride ions (H⁻) (hence, six electrons) are transferred to three molecules of
nicotinamide adenine dinucleotide (NAD1)

- One pair of hydrogen atoms (hence, two electrons) is transferred to one molecule of
flavin adenine dinucleotide (FAD).

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  Citrate Synthase Reaction
The first reaction of the cycle is condensation of acetyl-CoA (C-2) with oxaloacetate
(C-4) to yield a citrate (C-6).

Oxaloacetate + Acetyl CoA + H2O  Citrate + CoA + H+

- This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed

by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA,
a molecule that is energy rich because it contains the thioester bond that originated
in acetyl CoA.

1. Aconitase
The isomerization of citrate to isocitrate by aconitase is stereospecific, with the
migration of the -OH from the central carbon of citrate (C3) to C2. The
stereospecific nature of the isomerization determines that the CO2 lost, as
isocitrate is oxidized to succinyl-CoA.

Citrate  cis-aconitase + H2O

Cis-aconitase + H2O  isocitrate

2. Isocitrate Dehydrogenase
-ketoglutarate by isocitrate
dehydrogenase, (IDH).

Isocitrate + NAD  α-ketoglutarate CO2 + NADH

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 3. α-Ketoglutarate Dehydrogenase Complex
α-ketoglutarate is oxidatively decarboxylated to succinyl-CoA by the
α-ketoglutarate dehydrogenase (α-KGDH) complex.
This reaction generates the second Krebs cycle equivalent of CO2 and NADH.

α-ketoglutarate + NAD + CoA  succinyl-CoA + NADH + CO2

4. Succinyl CoA Synthetase (Succinyl Thiokinase )

The conversion of succinyl-CoA to succinate is catalysed by succinyl CoA synthetase

Succinyl CoA + Pi + GDP  succinate + GTP + CoA

In this process a high energy enzyme--phosphate intermediate is formed, with the
phosphate subsequently being transferred to GDP.
Mitochondrial GTP is used in a trans-phosphorylation reaction catalyzed by the
mitochondrial enzyme nucleoside diphospho kinase to phosphorylate ADP,
producing ATP and regenerating GDP for the continued operation of succinyl CoA
synthetase.

GTP + ADP  GDP + ATP

5. Succinate Dehydrogenase (SDH)

Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate.

Succinate + FAD  fumarate + FADH2

6. Fumarase (fumarate hydratase)

The fumarase-catalyzed reactions specific for the trans form of fumarate. The
result is that the hydration of fumarate proceeds stereospecifically with the
production of L-malate.

Fumarate + H2O  L-malate

7. Malate Dehydrogenase (MDH)

L-malate is the specific substrate for MDH, the final enzyme of the Krebs cycle.
The forward reaction of the cycle, the oxidation of malate yields oxaloacetate
(OAA).
Malate + NAD  oxaloacetate +NADH
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 The Citric Acid Cycle Is a Source of Biosynthetic Precursors

All 20 of the amino acids, excepting leucine and lysine, can be

degraded to Krebs cycle intermediates.

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 - Beriberi, a neurologic and cardiovascular disorder, is caused by a dietary deficiency
of thiamine (vitamin B1).
Your body needs thiamin to break down and digest the foods you eat, to keep your
metabolism going, and help your muscles and nervous system do their jobs
effectively.

The disease has been and continues to be a serious health problem in the Far East
because rice, the major food, has a rather low content of thiamine. The problem is
exacerbated if the rice is polished, because only the outer layer contains significant
amounts of thiamine.

Beriberi is also occasionally seen in alcoholics who are severely malnourished and
thus thiamine deficient. The disease is characterized by neurologic and cardiac
symptoms. Damage to the peripheral nervous system is expressed as pain in the
limbs, weakness of the musculature, and distorted skin sensation. The heart may be
enlarged and the cardiac output inadequate.

Which biochemical processes might be affected by a deficiency of thiamine?

Thiamine pyrophosphate is the prosthetic group of three important enzymes:
pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase.

Transketolase functions in the pentose phosphate pathway, which will be discussed

later. The common feature of enzymatic reactions utilizing TPP is the transfer of an
activated aldehyde unit.

In beriberi, the levels of pyruvate and α-ketoglutarate in the blood are higher than
normal. The increase in the level of pyruvate in the blood is especially pronounced
after the ingestion of glucose. A related finding is that the activities of the pyruvate
and α-ketoglutarate dehydrogenase complexes in vivo are abnormally low.

Why does TPP deficiency lead primarily to neurological disorders?

The nervous system relies essentially on glucose as its only fuel. In contrast, most
other tissues can use fats as a source of fuel for the citric acid cycle. The product of
aerobic glycolysis, pyruvate, can enter the citric acid cycle only through the
pyruvate dehydrogenase complex.
Symptoms similar to those of beriberi arise if an organism is exposed to mercury or
arsenite.
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  Oxidative Phosphorylation (Production of ATP)
Oxidative phosphorylation is the process in which ATP is formed as a result of the
transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers.

- The NADH and FADH2 formed in glycolysis, fatty acid oxidation, and the citric acid
cycle are energy-rich molecules because each contains a pair of electrons having a
high transfer potential.

- When these electrons are used to reduce molecular oxygen to water, a large
amount of free energy is liberated, which can be used to generate ATP.

- This process, which takes place in mitochondrial inner membrane, is the major
source of ATP in aerobic organisms.

- The flow of electrons from NADH or FADH2 to O2 through protein complexes located
in the mitochondrial inner membrane leads to the pumping of protons out of the
mitochondrial matrix.

- The resulting uneven distribution of protons generates a pH gradient and a

transmembrane electrical potential that creates a proton-motive force.

- ATP is synthesized when protons flow back to the mitochondrial matrix through an
enzyme complex.

- Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton
gradient across the inner mitochondrial membrane

- Oxygen is the final electron acceptor in this process.

- NADH is a powerful reducing agent  NADH is oxidized to NAD+

- O2 is a powerful oxidizing agent  oxygen is reduced to water.

- The outer membrane of mitochondria is more permeable than the inner membrane.

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 FAD = flavin adenine dinucleotide

NAD+ NADH

https://www.youtube.com/watch?app=desktop&v=LQmTKxI4Wn4

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 - In this simple version of the chemical osmotic theory applied to mitochondria,
electrons from NADH and other oxidizable substrates pass through a chain of
carriers (cytochromes, etc.) arranged asymmetrically in the membrane. Proton
transfer accompanies electron flow across the mitochondrial membrane, producing
both a chemical (∆pH) and an electrical gradient.

- The inner mitochondrial membrane is impermeable to protons; proton can enter

the matrix only through proton-specific channels (Fo). The proton-motive force that
drives protons back into the matrix provides the energy for ATP synthesis, catalyzed
by the F1 complex associated with Fo.

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 - Succinate-Q reductase, contains the succinate dehydrogenase that generates FADH2
in the citric acid cycle.

- Succinate-Q reductase, in contrast with the other complexes, does not pump
protons.

- NADH-Q oxidoreductase, succinate-Q reductase, Q-cytochrome c oxidoreductase,

and cytochrome c oxidase are also called Complex I, II, III, and IV respectively.

- Ubiquinones, or coenzyme Q (Q): Small non-protein carriers.

Q accept and release protons and electrons.

NADH enters complex I  III  IV.

FADH2 enters complex II  III  IV.

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 - The Fo and F1 subunits are connected in two ways, by the central γ ε stalk and by an
exterior column (α subunit, two β subunits, and the δ subunit).

- Fo is present inside the mitochondrial membrane.

- F1 is present inside the mitochondria.

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 - An uncoupling protein (UCP) is a mitochondrial inner membrane protein that is a
regulated proton channel or transporter.

There are 3 types: UCP-1, UCP-2, and UCP-3.

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  The overall stoichiometry of the Krebs cycle is:

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O

 2CO2 + 3NADH + FADH2 + GTP + 2H+ + HSCoA

3NADH = 3x3ATP = 9ATP

FADH = 2ATP
GTP = ATP
Total = 12 ATP per Acetyl-CoA
12ATP x2 = 24 ATP per 2 Acetyl-CoA

- Total energy yield of Glucose oxidation:

1) Glycolysis: 8 ATP
2) Pyruvate + CoA + NAD+  CO2 + acetyl-CoA + NADH = 2 x 3ATP = 6ATP
3) Krebs cycle 24 ATP

- Electron transport chain yields 34 ATP molecules for every 1 mole of glucose.

- A total of 36 ATPs are gained by complete oxidation of glucose.

- During glycolysis, a total 4 ATPs are formed out of which 2 are consumed.
Conversion of pyruvate to Acetyl CoA does produce any ATP. However, the 2
molecules of NADH + H+ are produced per glucose, which will yield 06 ATP in the
ETC.

- The Krebs cycle produces two molecules of ATP for every molecule of glucose. The
Krebs cycle also produces eight molecules of NADH and two molecules of FADH2 per
molecule of glucose. As each molecule of NADH is equivalent to three ATPs and
each FADH2 is equal to two ATPs, we get 2+ 8×3 + 2×2 = 30 ATPs from Krebs cycle.

- 2 ATP is required to transport NADH + H+ formed by glycolysis from the cytoplasm

through the inner mitochondrial membrane. Since 2 ATP are used, therefore only 36
ATP are produced instead of 38 ATP.

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 ATP yield from the complete oxidation of glucose
When considering:
NADH = 2.5 ATP
FADH2 = 1.5 ATP

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 Chapter 9
Glycogen

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  Introduction
- Glucose cannot be stored, because high concentrations of glucose disrupt the
osmotic balance of the cell, which would cause cell damage or death. How can
adequate stores of glucose be maintained without damaging the cell?
The solution to this problem is to store glucose as a nonosmotically active polymer
called glycogen.

- Stores of readily available glucose to supply the tissues with an oxidizable energy
source are found principally in the liver, as glycogen.

- A second major source of stored glucose is the glycogen of skeletal muscle.

However, muscle glycogen is not generally available to other tissues, because
muscle lacks the enzyme glucose-6-phosphatase.

- The major site of daily glucose consumption (75%) is the brain via aerobic pathways.
Most of the remainder of is utilized by erythrocytes, skeletal muscle, and heart
muscle.

- The body obtains glucose either directly from the diet or from amino acids and
lactate via gluconeogenesis.

- Glucose obtained from these two primary sources either remains soluble in the
body fluids or is stored in a polymeric form, glycogen.

- Glycogen is considered the principal storage form of glucose and is found mainly in
liver and muscle, with kidney and intestines adding minor storage sites. With up to
10% of its weight as glycogen, the liver has the highest specific content of any body
tissue.

- Muscle has a much lower amount of glycogen per unit mass of tissue, but since the
total mass of muscle is so much greater than that of liver, total glycogen stored in
muscle is about twice that of liver.

- Stores of glycogen in the liver are considered the main buffer of blood glucose
levels.

- Glycogen is polymer of α-glucose.

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 Glycogen - the glucose polymer

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 - Major pathways in carbohydrate metabolism
In animals, excess glucose is converted to its storage form, glycogen, by
glycogenesis. When glucose is needed as a source of energy (low-blood glucose
levels) or as a precursor molecule in biosynthetic processes, glycogen is degraded by
glycogenolysis.

Glucose can be converted to ribose-5-phophate (a component of nucleotides) and

NADPH (a powerful reducing agent) by means of the pentose phosphate pathway.
Glucose is oxidized by glycolysis, an energy-generating pathway that converts it to
pyruvate.

In the absence of oxygen, pyruvate is converted to lactate. When oxygen is present,

pyruvate is further degraded to form acetyl-CoA.
Significant amount of energy in the form of ATP can be extracted from acetyl-CoA
by the citric acid cycle and the electron transport system.

Note that carbohydrate metabolism is inextricably linked to the metabolism of the

nutrients. For example, acetyl-CoA is also generated from the breakdown of fatty
acids and certain amino acids. When acetyl-CoA is present in excess, a different
pathway converts it into fatty acids.

- Glycogen is not as reduced as fatty acids (fat contain more electrons) are and
consequently not as energy rich.
Why isn’t all excess fuel stored as fatty acids rather than as glycogen?

The controlled release of glucose from glycogen maintain blood-glucose levels

between meals. The circulating blood keeps the brain supplied with glucose, which
is virtually the only fuel used by the brain, except during prolonged starvation.
Moreover, the readily mobilized glucose from glycogen is a good source of energy
for sudden, strenuous activity. Unlike fatty acids, the released glucose can provide
energy in the absence of oxygen and can thus supply energy for anaerobic activity.

- Although most tissues have some glycogen, the two major sites of glycogen storage
are the liver and skeletal muscle. The concentration of glycogen is higher in the liver
than in muscle (10% versus 2% by weight), but more glycogen is stored in skeletal
muscle overall because of muscle’s much greater mass.

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 - Glycogen is present in the cytoplasm with the molecule appearing as granules.
In the liver, glycogen synthesis and degradation are regulated to maintain blood-
glucose levels as required to meet the needs of the organism as a whole. In
contrast, in muscle, these processes are regulated to meet the energy needs of the
muscle itself.

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  Glycogen synthesis
The glucose-6-phosphate is converted to glucose-6-phosphate by
phosphoglucomutase.
We transfer the phosphate group from C6 to C1 because UDP is going to bind to the
glucose.

PPi 2 Pi 2 phosphate groups are released

Specifically pyrophosphate (PPi)

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 The formation of glycogen from UDP-glucose (Uridine diphosphate glucose) requires
two enzymes:
a. Glycogen synthase
Which catalyzes the transfer of the glucosyl group of UDP-glucose to the
nonreducing ends of glycogen.

b. Amylo-α(1,4  1,6)-glucosyl transferase (branching enzyme)

Which creates the α-(1,6) linkages for branches in the molecule.
Catalyzes the transfer of a terminal fragment of 6-7 glucose residues from a
nonreducing end to the C-6 hydroxyl group of a glucose residue deeper into
the interior of the glycogen molecule.

Glycogen synthesis requires a preexisting tetrasaccharide composed of four α(1,4)-

linked glucosyl residues. The first of these residues is linked to a specific tyrosine
residue in a “primer” protein called glycogenin. The glycogen chain is then extended
by glycogen synthase and branching enzyme.

Was UDP glucose

T reducing bind with

non-reducing

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 Glycogenin
- Glycogen biosynthesis involves a specific initiation event, mediated by a specialized
protein, glycogenin.

Glycogenin has the unusual property of catalyzing its own glycosylation, attaching
C-1 of a UDP-glucose to a tyrosine residue on the enzyme. The attached glucose is
believed to serve as the primer required by glycogen synthase.

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  Glycogen degradation (Glycogenolysis)
- Degradation of stored glycogen (glycogenolysis) occurs through the action of
glycogen phosphorylase (most prominent enzyme of this process).

- The action of phosphorylase is to phosphorolytically remove single glucose residues

from α-(1,4)-linkages (nonreducing ends) within the glycogen molecules (the ends
with a free OH group on carbon 4)

- The product of this reaction is glucose-1-phosphate. The advantage of the reaction

proceeding through a phosphorolytic step is that:

1. The glucose is removed from glycogen is an activated state, i.e. phosphorylated

and this occurs without ATP hydrolysis.

2. The concentration of Pi in the cell is high enough to drive the equilibrium of the
reaction to the favorable direction since the free energy change of the standard
state reaction is positive, substantially favoring phosphorolysis.

Reversible

- The reaction catalyzed by phosphorylase is readily reversible in vitro. At pH 6.8, the

equilibrium ratio of orthophosphate to glucose 1-phosphate is 3.6. The value of ∆G0 ҆
for this reaction is small because a glycosidic bond is replaced by a phosphoryl ester
bond that has a nearly equal transfer potential.

- The phosphorolytic cleavage of glycogen is energetically advantageous because the

released sugar is already phosphorylated. In contrast, a hydrolytic cleavage would
yield glucose, which would then have to be phosphorylated at the expense of a
molecule of ATP to enter the glycolytic pathway.
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 - The glucose-1-phosphate produced by the action of phosphorylase is converted to
glucose-6-phosphate by phosphoglucomutase:
This enzyme contains a phosphorylated amino acid in the active site. The enzyme
phosphate is transferred to C-6 of glucose-1-phosphate generating glucose-1,6
phosphate as an intermediate.
The phosphate on C-1 is then transferred to the enzyme regenerating it and glucose-
6-phospahte is the released product.

- As mentioned above the phosphorylase mediated release of glucose from glycogen

yields a charged glucose residue without the need for hydrolysis of ATP.

- An additional necessity of releasing phosphorylated glucose from glycogen ensures

that the glucose residues do not freely diffuse from the cell. In the case of muscle
cells, this is acutely apparent since the purpose in glycogenolysis in muscle cells is to
generate substrate for glycolysis.

- The conversion of glucose-6-phosphate to glucose, which occurs in the liver, kidney

and intestine, by the action of glucose-6-phosphatase.

- Skeletal muscle as these cells lack glucose-6-phosphatase. Therefore, any glucose

released from glycogen stores of muscle will be oxidized in the glycolytic pathway.

- In the liver, the action of glucose-6-phosphatase allows glycogenolysis to generate

free glucose for maintaining blood glucose levels.

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 The most abundant

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  Debranching Enzyme
- Glycogen phosphorylase acting alone degrades glycogen to a limited extent.
Glycogen phosphorylase cannot remove glucose residues from the branch points
(alpha-1,6 linkages) in glycogen.

- Indeed, phosphorylase stops cleaving α-1,4 linkages when it reaches a terminal

residue four residues away from a branch point.
Because about 1 in 10 residues is branched, cleavage by the phosphorylase alone
would come to a halt after the release of six glucose molecules per branch.

- The removal of the branch point glucose residues requires the action of debranching
enzyme (also called glucan transferase) which contains 2 activities:
1. Glucotransferase
The transferase shifts a block of three glucosyl residues from one outer branch
to another. This transfer exposes a single glucose residue joined by an α-1,6-
glycosidic linkage.

2. Glucosidase
Also known as the α-1,6-Glucosidase, hydrolyzes the α-1,6-glycosidic bond.

- The steps (picture below):

1. First, α-1,4-glycosidic bonds on each branch are cleaved by phosphorylase,
leaving four residues along each branch.
2. The transferase shifts a block of three glycosyl residues from one outer branch
to the other.
3. In this reaction, the α-1,4-glycosidic link between the blue and the green
residues is broken and a new α-1,4 link between the blue and the yellow
residues is formed.
4. The green residue is then removed by α-1,6-glucosidase, leaving a linear chain
with all α-1,4 linkages, suitable for further cleavage by phosphorylase.

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 - This glucose residue is uncharged since the glucosidase-catalyzed reaction is not
phosphorylytic. This means that theoretically glycogenolysis occurring in skeletal
muscle could generate free glucose that could enter the blood stream. However, the
activity of hexokinase in muscle is so high that any free glucose is immediately
phosphorylated by the lycolytic enzyme hexokinase, and enters the glycolytic
pathway. Indeed, the precise reason for the temporary appearance of the free
glucose from glycogen is the need of the skeletal muscle cell to generate energy
from glucose oxidation, thereby, precluding any chance of the glucose entering the
blood.

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  Regulation of Glycogenolysis  Glycogen Phosphorylase
- Regulation  Allosteric  local  such as ATP/Glucose
 Phosphorylation  Global  such as insulin/Glucose

- Distribution  Muscle
 Liver

- Glycogen metabolism is precisely controlled by multiple interlocking mechanisms.

The focus of this control is the enzyme glycogen phosphorylase.
Phosphorylase is regulated by several allosteric effectors that signal the energy
state of the cell as well as by reversible phosphorylation, which is responsive to
hormones such as insulin, epinephrine, and glucagon.

- Glycogen phosphorylase Is an enzyme that exist in two distinct conformational

states:
1. T (for tense, less active).
2. R (for relaxed, more active) state: can bind glycogen.

- The active R state

1. Is enhanced by binding of AMP.
2. Inhibited by binding ATP or glucose-6-phosphate.

- Glycogen phosphorylase is also subject to covalent modification by phosphorylation

as a means of regulating its activity. The relative activity of the un-modified
phosphorylase enzyme (given the name phosphorylase-b) is sufficient to generate
enough glucose-1-phosphate for entry into glycolysis for the production of sufficient
ATP to maintain the normal resting activity of the cell. This is true in both liver and
muscle cells.

- The phosphorylated phosphorylase-a is much more active than the b form

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 - Under most physiological conditions, phosphorylase b is inactive because of the
inhibitory effects of ATP and glucose-6-phosphate. In contrast, phosphorylase a is
fully active, regardless of the levels of AMP, ATP, and glucose-6-phosphate.
In resting muscle, nearly all enzyme is in the inactive b form.

- Phosphorylase b is converted into phosphorylase a by the phosphorylation of a

single serine residue (serine 14) in each subunit. This conversion is initiated by
hormones. Fear or the excitement of exercise will cause levels of the hormone
epinephrine of muscle result in phosphorylation of the enzyme to the
phosphorylase a form. The regulatory enzyme phosphorylase kinase catalyzes this
covalent modification.

- When ATP in muscle decreases & AMP increases T is converted to R state.

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 - Liver phosphorylase produces glucose for use by other tissues.
The role of glycogen degradation in the liver is to form glucose for export to other
tissues when the blood-glucose level is low. Consequently, we can think of the
default state of liver phosphorylase as being the a form: glucose is to be generated
unless the enzyme is signaled otherwise.

The liver phosphorylase a form thus exhibits the most responsive R  T

transition.
The binding of glucose shifts the a form from the active R state to the less-active T.
In essence, the enzyme reverts to the low-activity T state only when it detects the
present of significant glucose. If glucose is present in the diet, there is no need to
degrade glycogen. As we will see later, the presence of glucose also facilitates the a-
to-b transition.

- Conformational changes occurs to convert the enzyme to more active or less active.

- The regulation of liver phosphorylase differs from that of muscle phosphorylase. In

muscle, the default state is the b form: there is no need to generate glucose unless
energy is required.
As discussed previously, AMP shifts the muscle B form from T to the R state. Unlike
the enzyme in the muscle, the liver phosphorylase is insensitive to regulation by
AMP because the liver doesn’t undergo the dramatic changes in energy charge seen
in contracting muscle.
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 - In human beings, liver phosphorylase and muscle phosphorylase are approximately
90% identical in amino acid sequence, yet the 10% difference results in subtle but
important shifts in the stability of various forms of the enzyme.

 Low Glucose levels

In response to lowered blood glucose the in the liver
- Alpha cells of the pancreas secrete glucagon which binds to cell surface receptors on
liver and several other cells. Liver cells are the primary target for the action of this
peptide hormone. The response of cells to the binding of glucagon to its cell surface
receptor is
1. The activation of the enzyme adenylate cyclase which is associated with the
receptor. Activation of adenylate cyclase leads to a large increase in the
formation of cAMP.
2. cAMP binds to an enzyme called cAMP-dependent protein kinase, PKA. Binding
of cAMP to the regulatory subunits of PKA leads to the release and subsequent
activation of the catalytic subunits.
3. The catalytic subunits then phosphorylate a number of proteins on serine and
threonine residues. Of significance to this discussion is the PKA-mediated
phosphorylation of phosphorylase kinase.
4. Phosphorylation of phosphorylase kinase activates the enzyme, which in turn
phosphorylates the b form of phosphorylase. Phosphorylation of phosphorylase-
b greatly enhances its activity towards glycogen breakdown.
5. The modified enzyme is called phosphorylase-a. The net result is an extremely
large induction of glycogen breakdown in response to glucagon binding to cell
surface receptors.

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 In response to lowered blood glucose the in the Muscle
- This identical cascade of events occurs in skeletal muscle cells as well. However, in
these cells the induction of the cascade is the result of epinephrine binding to
receptors on the surface of muscle cells. Epinephrine is released from the adrenal
glands in response to neural signals indicating an immediate need for enhanced
glucose utilization in muscle, the so called fight or flight response.
- Muscle cells lack glucagon receptors.
- Regulation of phosphorylase kinase activity is also affected by two distinct
mechanisms involving Ca2+ ions.
- The ability of Ca2+ ions to regulate phosphorylase kinase is through the function of
one of the subunits of this enzyme. One of the subunits of this enzyme is the
ubiquitous protein, calmodulin. Calmodulin is a calcium binding protein.
- Binding induces a conformational change in calmodulin which in turn enhances the
catalytic activity of the phosphorylase kinase towards its substrate, phosphorylase-
b. This activity is crucial to the enhancement of glycogenolysis in muscle cells where
muscle contraction is stimulated acetylcholine stimulation of neuromuscular
junctions. The effect of acetylcholine release from nerve terminals at a
neuromuscular junction is to depolarize the muscle cell leading to increased release
of sarcoplasmic Ca2+, thereby activating phosphorylase kinase. Thus, not only does
the increased intracellular calcium increase the rate of muscle contraction it
increases glycogenolysis which provides the muscle cell with the increased ATP it
also needs for contraction.

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  ‫شرح العمليتين بطريقة واحدة من ساليدات الدكتورة ِصبا‬
- The signal molecules epinephrine and glucagon bind to specific seven-
transmembrane (7TM) receptors in the plasma membrane of target cells.
- The GTP-bound subunit of Gs activiates the transmembrane protein adenylate
cyclase. This enzyme catalyzed the formation of the second messenger cyclic AMP
from ATP.
- The elevated cytoplasmic level of cyclic AMP activates protein kinase A. The binding
of cyclic AMP to inhibitory regulatory subunits triggers their dissociation from
catalytic subunits. The free catalytic subunits are now active.
- Protein kinase A phosphorylates phosphorylase kinase first of β subunit and then on
the α subunit, which subsequently activates glycogen phosphorylase.
- The cyclic AMP cascade high amplifies the effect of hormones. The binding of small
number of hormone molecules to cell-surface receptors leads to the release of a
very large number of sugar units.

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 - Insulin inactivates glycogen synthase kinase
Insulin triggers a cascade that leads to the phosphorylation and inactivation of
glycogen synthase kinase and prevents the phosphorylation of glycogen synthase.
Protein phosphatase 1 (PP1) removes the phosphates from the glycogen synthase
thereby activating the enzyme and allowing glycogen synthesis.
IRS  Insulin-receptor substrate.

- Recall that protein kinase A adds a phosphoryl group to phosphorylase kinase,

activating that enzyme and initiating glycogen breakdown. Likewise, protein kinase
A adds a phosphoryl group to glycogen synthase, but this phosphorylation leads to a
decrease in its enzymatic activity.

- PP1 revers the regulatory effects of kinases on glycogen metabolism

PP1 inactivates phosphorylase a and phosphorylase kinase by dephosphorylating
them. PP1 decreases the rate of glycogen breakdown; it reveres the effect of the
phosphorylation cascade. Moreover, PP1 also removes phosphoryl groups from
glycogen synthase b to convert it into much more active glycogen synthase a form.
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 Glycogen synthase and glycogen synthase
kinase and opposite to each other

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 - The infusion of glucose into the bloodstream leads to the inactivation of
phosphorylase, followed by the activation of glycogen synthase, in the liver.

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 - Glucose binds to and inhibits glycogen phosphorylase a in the liver, facilitating the
formation of the T state of phosphorylase a. The T state of phosphorylase a does
not bind protein phosphatase 1 (PP1), leading to the dissociation and activation of
PP1 from glycogen phosphorylase a. The free PP1 dephosphorylates glycogen
phosphorylase a and glycogen synthase b, leading to the inactivation of glycogen
breakdown and the activation of glycogen synthesis.

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 Chapter 01
Lipids

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  Major Roles of Biological of Lipids
- Biological molecules that are insoluble in aqueous solutions and soluble in organic
solvents are classified as lipids.
The lipids of physiological importance for humans have four major functions:
1. Structural components of biological membranes  Phospholipids.
2. Energy reserves, predominantly in the form of triacylglycerols  Fat.
3. Precursors of many hormones  Steroids.
4. Precursors of bile acids; aid in lipid solubilization  Fatty acids.

 Triacylglycerides
Triacylglycerol is composed of one glycerol unit and three fatty acid chains, which can
vary in length and hydrogen saturation.
Fatty acids are long-chain hydrocarbon molecules containing a carboxylic acid moiety
at one end.

Carbon 15-20

‫بالعادة الرقم بكون زوجي‬

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 - The notation 18:0 denotes a C18 fatty acid with no double bonds, whereas 18:2
signifies that there are two double bonds.

Cys- ∆9

Carboxylic acid when bound to

another chain it is called acyl
50-80% of olive oil
At pH = 7

- The numbering of carbons in fatty acids begins with the carbon of the carboxyl
group.
- At physiological pH, the carboxyl group is ionized (COO-), causing a negative charge
onto fatty acids.
- Fatty acids that contain no carbon-carbon double bonds are termed saturated fatty
acids.
- Fatty acids that contain double bonds are unsaturated fatty acids.
- The numeric designations used for fatty acids come from the number of carbon
atoms, followed by the number of sites of unsaturation (eg, palmitic acid is a 16-
carbon fatty acid with no unsaturation and is designated by 16:0).
- The site of unsaturation in a fatty acid is indicated by the symbol ∆ and the number
of the first carbon of the double bond (e.g. palmitoleic acid is a 16- carbon fatty acid
with one site of unsaturation between carbons 9 and 10, and is designated by
16:1∆9).

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 - Melting point decrease with increasing double bonds and decrease carbon numbers.

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 - Saturated fatty acids of less than eight carbon atoms are liquid at physiological
temperature.
- Saturated fatty acids of more than ten are solid at physiological temperature.
- The presence of double bonds in fatty acids significantly lowers the melting point
relative to a saturated fatty acid.
- The majority of body fatty acids are acquired in the diet. However, the lipid
biosynthetic capacity of the body (fatty acid synthase and other fatty acid modifying
enzymes) can supply the body with all the various fatty acid structures needed.
- Mammals lack the enzymes to introduce double bonds at carbons beyond C- 9 in
the fatty acid chain. Therefore two fatty acid are essential:
1. linoleic acid
2. linolenic acid

- These two fatty acids contain unsaturation sites beyond carbons 9 and 10. Linoleic
acid and linolenic acid cannot be synthesized from precursors in the body and are
thus considered the essential fatty acids; essential in the sense that they must be
provided in the diet. Since plants are capable of synthesizing linoleic and linolenic
acid humans, can acquire these fats by consuming a variety of plants or else by
eating the meat of animals that have consumed these plant fats.

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 Chapter 01
Fatty acid Metabolism

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  Major physiological roles:
1. Building blocks of phospholipids and glycolipids.
2. Can covalently attach proteins targeted to membranes.
3. Hormones and intracellular messengers.
4. Fuel molecules (carries more electrons than glycogen).
The yield from the complete oxidation of fatty acids is about 9 kcal g-1.about 4 kcal
g-1 (17 kJ g-1) for carbohydrates and proteins.

- Migratory birds (American golden plover), can fly great distances without eating.
From Alaska to the southern tip of South America; a large segment of the flight
=3800 km. Imagine what would be the bird weigh if his fuel was glycogen!!

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 - Fatty acid degradation occurs in the mitochondria (β-oxidation).
Beta-oxidation is the catabolic process by which fatty acid molecules are broken in
the mitochondria to generate acetyl-CoA.

- Fatty acid synthesis occurs in the cytosol.

- Number of acetyl groups

produced = n/2

- Number of degradation of
n carbon compound =
𝒏
-1
𝟐

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  Highly concentrated store of energy
1. Reduced
2. Anhydrous (contains no water)
- Triacylglycerols are nonpolar, and so they are stored in a nearly anhydrous form,
whereas much more polar carbohydrates are more highly hydrated.

- In fact, 1 g of dry glycogen binds about 2 g of water. Consequently, a gram of nearly

anhydrous fat stores 6.75 times as much energy as a gram of hydrated glycogen,
which is likely the reason that triacylglycerols rather than glycogen were selected in
evolution as the major energy reservoir.

- 9gm of glycogen = 4gm of fat

But 1gm glycogen binds with 2gm water  so 9gm glycogen = 27gm total.
27gm glycogen gives energy as 4gm fat.
That is why we prefer fat on glycogen.

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 - Cells that use fatty acids for energy get them from 3 sources:
1. Diet
2. Stored Fats
3. In animals, fatty acids can be synthesized

- Mammals lack the enzymes to introduce double bonds at carbons beyond C-9 in the
fatty acid chain. Therefore two fatty acid are essential:
1. linoleic acid
2. linolenic acid

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249 | P a g e
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  Fatty acid metabolism
- Utilization of dietary lipids requires that they first be absorbed through the
intestine.

- As these molecules are oils, they would be essentially insoluble in the aqueous
intestinal environment.

- Solubilization (emulsification) of dietary lipid is accomplished via bile salts,

amphipathic that are synthesized in the liver and secreted from the gallbladder.

- The emulsified fats can then be degraded by pancreatic lipases (lipase and
phospholipase A2). These enzymes, secreted into the intestine from the pancreas
generate free fatty acids and a mixture of mono- and diacylglycerols from dietary
triacylglycerols.

- These digestion products are carried in micelles to the intestinal epithelium where
they are absorbed across the plasma membrane.

- Following absorption of the products of pancreatic lipase by the intestinal mucosal

cells, the re synthesis of triacylglycerols occurs.

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 - The triacylglycerols are then solubilized in lipoprotein complexes (complexes of lipid
and protein) called chylomicrons.
A chylomicron contains lipid droplets surrounded by the more polar lipids and
finally a layer of proteins.

These particles are composed mainly of triacylglycerols, with apoprotein B- 48 as

the main protein component.
Protein constituents of lipoprotein particles are called apolipoproteins.
Chylomicrons also function in the transport of fat-soluble vitamins and cholesterol.

- Triacylglycerols synthesized in the liver are packaged into VLDLs and released into
the blood directly.

- Chylomicrons from the intestine are then released into the blood via the lymph
system for delivery to the various tissues for storage or production of energy
through oxidation.

- The triacylglycerol components of VLDLs and chylomicrons are hydrolyzed to free

fatty acids and glycerol in the capillaries of adipose tissue and skeletal muscle by
the action of lipoprotein lipase.

- The free fatty acids are then absorbed by the cells and the glycerol is returned via
the blood to the liver (and kidneys). The glycerol is then converted to the glycolytic
intermediate DHAP.

- The classification of blood lipids is distinguished based upon the density of the
different lipoproteins. As lipid is less dense than protein, the lower the density of
lipoprotein the less protein there is.
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252 | P a g e
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 - Glucagon and epinephrine activate
glycolysis.

- Glycerol is hydrophilic, therefore it

doesn’t need transport.

- Fatty acid is hydrophobic, therefore

it needs transport.

- Adipose cells are specialized for the

synthesis and storage of
triacylglycerols and for their
mobilization into fuel molecules
that are transported to other
tissues by the blood.

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  Utilization of Fatty Acids as Fuel Requires Three Stages of Processing
1. The lipids must be mobilized. In this process, triacylglycerols are degraded to fatty
acids and glycerol, which are released from the adipose tissue and transported to
the energy-requiring tissues
2. The fatty acids must be activated and transported into mitochondria for
degradation.
3. The fatty acids are broken down in a step-by step fashion into acetyl CoA, which is
then processed in the citric acid cycle.

- Triacylglycerol are hydrolyzed by hormone-stimulated lipases

Consider someone who just awakened from a night’s sleep and begins a bout of
exercise. Glycogen stores will be low, but lipids are readily available. How are these
lipid stores mobilized?

Before fats can be used as fuels, the triacylglycerol storage form must be hydrolyzed
to yield isolated fatty acids. This reaction is catalyzed by a hormonally controlled
lipases. Under physiological conditions facing an early-morning runner, glucagon
and epinephrine will be present. In adipose tissue, these hormones trigger 7TM
receptors that activate adenylate cyclase. The increases level of cyclic stimulated
PKA. PKA phosphorylate lipases.

7TM receptors  adenylate cyclase  cAMP  PKA  phosphorylation of lipases.

- FA are not soluble in blood plasma, serum albumin binds the FA and serves as a
carrier.
- Insulin inhibits breakdown of fat in adipose tissue by inhibiting the intracellular
lipase that hydrolyzes triglycerides to release fatty acids.
- Insulin stimulates fatty acid synthesis in white and brown fat cells as well as in liver
and mammary tissue (storage).

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 Doesn’t need activation, when MAG
arrives it works

Hormone
sensitive (HS)

Can break TAG

but works on DAG

When phosphorylated it activate ATGL

Adipose Triglyceride Lipase (ATGL)

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 - Glycerol is absorbed by the liver and phosphorylated, oxidized to dihydroxyacetone
phosphate, and then isomerized to glyceraldehyde 3-phosphate, an intermediate in
both the glycolytic and the gluconeogenic pathways.

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  Fatty acid β-Oxidation
- The primary sources of fatty acids for oxidation are dietary and mobilization from
cellular stores.

- Fatty acids from the diet can are delivered from the gut to cells via transport in the
blood. Fatty acids are stored in the form of triacylglycerols primarily within
adipocytes of adipose tissue.

- In response to energy demands, the fatty acids of stored triacylglycerols can be

mobilized for use by peripheral tissues.

- The release of metabolic energy, in the form of fatty acids, is controlled by a

complex series of interrelated cascades that result in the activation of hormone
sensitive lipase.

- The stimulus to activate this cascade, in adipocytes, can be

1. Glucagon
2. Epinephrine
3. Β-corticotropin

- The process of fatty acid oxidation is termed β-oxidation since it occurs through the
sequential removal of 2-carbon units by oxidation at the β- carbon position of the
fatty acyl-CoA molecule.
- Each round of β-oxidation produces one mole of NADH, one mole of FADH2 and one
mole of acetyl-CoA.
- The acetyl-CoA, the end product of each round of β-oxidation, then enters the TCA
cycle, where it is further oxidized to CO2 with the concomitant generation of three
moles of NADH, one mole of FADH2 and one mole of ATP. The NADH and FADH2
generated during the fat oxidation and acetyl-CoA oxidation in the TCA cycle then
can enter the respiratory pathway for the production of ATP.

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 1. Step 1  Activation
This process occurs on the outer mitochondrial membrane.
Activation is catalyzed by acyl-CoA synthetase (also called fatty acid thiokinase/ fatty
acyl-CoA ligase).

The net result of this activation process is the consumption of 2 ATP.

- Sum

- One high-transfer-potential compound is cleaved (between PPi and AMP) and one
high-transfer-potential compound is formed (the thioester acyl CoA)
- Equilibrium constant (Kc) = 1.

- How is the overall reaction driven forward?

Pyrophosphate is rapidly hydrolyzed by a pyrophosphatase, and so the complete
reaction is:

- Many biosynthetic reactions are made irreversible by the hydrolysis of inorganic

pyrophosphate.

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 2. Step 2  Transport into matrix
Activated long-chain fatty acids are transported across the membrane by conjugating
them to carnitine, a zwitterionic alcohol. This reaction is catalyzed by carnitine
acyltransferase I (also called carnitine palmitoyl transferaseI), which is bound to the
outer mitochondrial membrane.

A number of diseases have been traced to a deficiency of carnitine, the transferase

or the translocase. The symptoms of carnitine deficiency range from mild muscle
cramping to severe weakness and even death. The muscle, kidney, and heart are
the tissues primarily affected. Muscle weakness during prolonged exercise is an
important characteristic of a deficiency of carnitine acyl transferases because
muscle relies on fatty acids as a long-term source of energy.

Acyl  8-10 carbon can pass through mitochondrial membrane without translocase.
Acyl above 10 carbons need translocase.

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 3. Step 3  Degradation of fatty acid
Fatty acids are degraded by the repetition of a four-reaction sequence consisting of:
oxidation, hydration, oxidation, and thiolysis.

β oxidation pathway of a saturated acyl CoA

1) Oxidation by FAD
2) Hydration
3) Oxidation by NAD+
4) Thiolysis by CoA

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 1) Oxidation by FAD
As in the dehydrogenation of succinate in the citric acid cycle, FAD rather than
NAD+ is the electron acceptor because the ΔG for this reaction is insufficient to
drive the reduction of NAD+.

2) Hydration

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 3) Oxidation by NAD+

4) Thiolysis (Cleavage)

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  First three rounds in the degradation of Palamitate (C16).
16 carbons  7 cycles  7 NADH – 7 FADH2 – H2O – 8 acetyl groups.

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 - The Complete Oxidation of Palmitate Yields 106 Molecules of ATP
The degradation of palmitoyl CoA (C16-acyl CoA) requires seven reaction cycles. In
the seventh cycle, the C4-ketoacyl CoA is thiolyzed to two molecules of acetyl CoA.
Hence, the stoichiometry of oxidation of palmitoyl CoA is:

- Another example
The net result of the oxidation of one mole of oleic acid (an 18-carbon fatty acid)
will be 146 moles of ATP (2 mole equivalents are used during the activation of the
fatty acid), as compared with 114 moles from an equivalent number of glucose
carbon atoms.
8 cycles: 8 x 5 ATP = 40 ATP
9 Acetyl-CoA x 12 ATP = 108 ATP
Subtotal 148ATP - 2 ATP used during the activation of the fatty acid
Total 146 mol ATP

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  Certain Fatty Acids Require Additional Steps for Degradation
- The β-oxidation pathway accomplishes the complete degradation of saturated fatty
acids having an even number of carbon atoms. Most fatty acids have such structures
because of their mode of synthesis.

- Consider the oxidation of palmitoleate. This C16 unsaturated fatty acid, which has
one double bond between C-9 and C- 10, is activated and transported across the
inner mitochondrial membrane in the same way as saturated fatty acids.
Palmitoleoyl CoA then undergoes three cycles of degradation, which are carried out
by the same enzymes as in the oxidation of saturated fatty acids. However, the cis-D
3-enoyl CoA formed in the third round is not a substrate for acyl CoA
dehydrogenase. The presence of a double bond between C-3 and C-4 prevents the
formation of another double bond between C-2 and C-3. This impasse is resolved by
a new reaction that shifts the position and configuration of the cis-D 3 double bond
by isomerase. The subsequent reactions are those of the saturated fatty acid
oxidation pathway, in which the trans-D 2-enoyl CoA is a regular substrate.

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 - Odd-numbered double bonds are handled by the isomerase, and even-numbered
ones by the reductase and the isomerase.

- Another problem arises with the oxidation of polyunsaturated fatty acids.

Consider linoleate, a C18 polyunsaturated fatty acid with cis-D 9 and cis-D 12 double
bonds. The cis-D 3 double bond formed after three rounds of β oxidation is
converted into a trans-D 2 double bond by the aforementioned isomerase. The acyl
CoA produced by another round of β-oxidation contains a cis-D 4 double bond.
Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl
intermediate, which is not a substrate for the next enzyme in the b-oxidation
pathway.
- This impasse is circumvented by 2,4-dienoyl CoA reductase, an enzyme that uses
NADPH to reduce the 2,4-dienoyl intermediate to trans-D 3-enoyl CoA. cis- D 3-
Enoyl CoA isomerase then converts trans-D 3-enoyl CoA into the trans-D 2 form, a
customary intermediate in the β-oxidation pathway. These catalytic strategies are
elegant and economical. Only two extra enzymes are needed for the oxidation of
any polyunsaturated fatty acid.

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 - Odd-chain fatty acids yield propionyl CoA in the final thiolysis step.
Fatty acids having an odd number of carbon atoms are minor species. They are
oxidized in the same way as fatty acids having an even number, except that
propionyl CoA and acetyl CoA, rather than two molecules of acetyl CoA are
produced in the final round of degradation.
The activated three-carbon unit in propionyl CoA enters the citric acid cycle after it
has been converted into succinyl CoA.

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  Fatty acids are also oxidized in peroxisomes
- FAD gives electrons directly to O2 and form H2O2 then produce H2O and O2

- Subsequent steps are identical with their mitochondrial counterparts, although they
are carried out by different isoforms of the enzymes.

- Initiation of Peroxisomal Fatty Acid Degradation. The first dehydration in the

degradation of fatty acids in peroxisomes requires a flavoprotein dehydrogenase
that transfers electrons to O2 to yield H2O2.– NOT to the electron transport chain!

- Fatty acid oxidation in these organelles, which halts at octanoyl CoA, may serve to
shorten long chains to make them better substrates of β-oxidation in mitochondria.

- Peroxisomes don’t function in patients with Zellweger syndrome. Liver, kidney, and
muscle abnormalities usually lead to death by age six.
The syndrome is caused by a defect in the import of enzymes into the peroxisomes.
Here we see a pathological condition resulting from an inappropriate cellular
distribution of enzymes.

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  Ketone Bodies Are Formed from Acetyl Coenzyme A When Fat Breakdown
Predominates.
The acetyl CoA formed in fatty acid oxidation enters the TCA only if fat and
carbohydrate degradation are appropriately balanced. e.g., depends on the
availability of oxaloacetate for the formation of citrate. [oxaloacetate] is lowered if
carbohydrate is unavailable or improperly utilized. Recall that oxaloacetate is
normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase

In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic

pathway and hence is unavailable for condensation with acetyl CoA. Under these
conditions, acetyl CoA is diverted to the formation of ketone bodies.
Acetoacetate, D-3-hydrocybutyrate and acetone are often referred to as ketone
bodies.

- The odor of acetone may be detected in the breath of a person who has a high level
of acetoacetate in the blood.

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 - Ketone bodies are formed from acetyl CoA when fat breakdown predominates.
The acetyl CoA formed in fatty acids enters the citric acid cycle only if fat and
carbohydrate degradation are appropriately balance. Acetyl CoA must combine with
oxaloacetate to gain entry to the citric acid cycle.
The availability of oxaloacetate, however, depends on an adequate supply of
carbohydrate. Recall that oxaloacetate is normally formed from pyruvate, the
product of glucose degradation in glycolysis. If carbohydrate is unavailable or
improperly utilized, the concentration of oxaloacetate is lowered and acetyl CoA
cannot enter the citric acid cycle. This dependency is the molecular basis of the
adage that fats burn in the flame of carbohydrates.

Utilization of
Acetoacetate as a Fuel.

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 - The major site of production of acetoacetate and 3-hydroxybutyrate is the liver
(mitochondria).
- Heart muscle and the renal cortex use acetoacetate in preference to glucose.
- In prolonged starvation, 75% of the fuel needs of the brain are met by ketone
bodies.
- The absence of insulin (diabetes) has two major biochemical consequences.
1) The liver cannot absorb glucose and consequently cannot provide oxaloacetate
to process fatty acid-derived acetyl CoA.
2) Insulin normally limit fatty acid mobilization by adipose tissue.
- The liver thus produces large amounts of ketone bodies, which are moderately
strong acids. The result is severe acidosis. The decrease in pH impairs tissue
function, most importantly in the central nervous system.

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 - Acetoacetate is converted into acetyl CoA into two steps
1. First, acetoacetate is activated by the transfer of CoA from succinyl CoA in a
reaction catalyzed by a specific CoA transferase.
2. Second, acetoacetyl CoA is cleaved by thiolase to yield two molecules of acetyl
CoA, which can then enter the citric acid cycle.
- The liver has acetoacetate available to supply to other organs because it lacks this
particular CoA trasferase.

- 3-Hydroxybutyrate requires an additional step to yield acetyl CoA. It is first oxidized

to produce acetoacetate, which is precesses as heretofore described, and NADH for
use in oxidative phosphorylation.

- Ketone bodies can be regarded as a water soluble, transportable form of acetyl

units.

- Fatty acids are released by adipose tissue and converted into acetyl units by the
liver, which then exports them as acetoacetate. As might be expected, acetoacetate
also has a regulatory role. High levels of acetoacetate in the blood signify an
abundance of acetyl units and lead to a decrease in the rate of lipolysis in adipose
tissue.

- Animals Cannot Convert Fatty Acids into Glucose

Acetyl CoA cannot be converted into pyruvate or oxaloacetate in animals

- Plants have two additional enzymes enabling them to convert the carbon atoms of
acetyl CoA into oxaloacetate

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  Fatty acid synthesis
- Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and NADPH
through the action of enzymes called fatty acid synthases.

- This process takes place in the cytoplasm of the cell.

- Most of the acetyl-CoA which is converted into fatty acids is derived from
carbohydrates via the glycolytic pathway.

- Citrate Carries Acetyl Groups from Mitochondria to the Cytosol for Fatty Acid
Synthesis:

- NADH is concomitantly converted into that of NADPH by this series of reactions.

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  Differences between fat degradation and synthesis
1. Synthesis takes place in the cytoplasm, in contrast with degradation, which takes
place primarily in the mitochondrial matrix.

2. Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups
of an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown
are covalently attached to the sulfhydryl group of coenzyme A.

3. The enzymes of fatty acid synthesis in higher organisms are joined in a single
polypeptide chain called fatty acid synthase. In contrast, the degradative enzymes
do not seem to be associated.

4. The growing fatty acid chain is elongated by the sequential addition of two-
carbon units derived from acetyl CoA. The activated donor of two- carbon units in
the elongation step is malonyl ACP. The elongation reaction is driven by the
release of CO2.

5. The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid
degradation are NAD+ and FAD.

6. Elongation by the fatty acid synthase complex stops on the formation of

palmitate (C16). Further elongation and the insertion of double bonds are carried
out by other enzyme systems.

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  Steps of Fatty acid synthesis
- Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA.

- This irreversible reaction is the committed step in fatty acid synthesis.

- The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase (the essential
regulatory enzyme for fatty acid metabolism), which contains a biotin prosthetic
group.

- The activated CO2 group in this intermediate is then transferred to acetyl CoA to
form malonyl CoA.

- A carboxybiotin intermediate is formed at the expense of the hydrolysis a molecule

of ATP.

- The carboxyl group of biotin is covalently attached to the ε amino group of a lysine
residue, as in pyruvate carboxylase and propionyl CoA carboxylase.

- Intermediates in Fatty Acid Synthesis Are Attached to an Acyl Carrier Protein (ACP).

- Specifically, they are linked to the sulfhydryl terminus of a phosphopantetheine

group, which is, in turn, attached to a serine residue of the acyl carrier protein.
- Both acyl carrier protein and CoA include phosphopantetheine as their reactive
units.
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 - The Elongation Cycle in Fatty Acid Synthesis.

- The enzyme system that catalyzes the synthesis of saturated long-chain fatty acids
from acetyl CoA, malonyl CoA, and NADPH is called the fatty acid synthase.

- The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP
and malonyl ACP.

- Acetyl transacylase and malonyl transacylase catalyze these reactions.

- Acetyl ACP and malonyl ACP react to form acetoacetyl ACP. The acylmalonyl ACP
condensing enzyme catalyzes this condensation reaction.

- Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer

other than acetyl units, though at a much slower rate. The synthesis of fatty acids
with an odd number of carbon atoms starts with propionyl ACP, which is formed
from propionyl CoA by acetyl transacylase.

- Although HCO3 - is required for fatty acid synthesis, its carbon atom does not
appear in the product. Rather, all the carbon atoms of fatty acids containing an even
number of carbon atoms are derived from acetyl CoA.

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277 | P a g e
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 - In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl
ACP to form a C6-b-ketoacyl ACP.

- This reaction is like the one in the first round, in which acetyl ACP condenses with
malonyl ACP to form a C4-b- ketoacyl ACP.

- Reduction, dehydration, and a second reduction convert the C6-b-ketoacyl ACP into
a C6-acyl ACP, which is ready for a third round of elongation.

- The elongation cycles continue until C16-acyl ACP is formed. This intermediate is a
good substrate for a thioesterase that hydrolyzes C16-acyl ACP  palmitate + ACP.
The thioesterase acts as a ruler to determine fatty acid chain length. The synthesis
of longer-chain fatty acids needs further reactions.
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 - A multifunctional enzyme complex in animals synthesizes fatty acids.

- Fatty acid synthase is overexpressed in some tumors, thus inhibitors are exciting
candidates as antitumor and also as antiobesity drugs.

 The Acetyl CoA carboxylase (catalyzes the committed step in fatty acid synthesis) is
controlled by three global signals glucagon, epinephrine, and insulin that correspond
to the overall energy status of the organism.

- Insulin stimulates fatty acid synthesis by activating the carboxylase, glucagon and
epinephrine have the reverse effect.

- The carboxylase is also inhibited by phosphorylation and allosterically stimulated by

citrate.

Activated by insulin by
dephosphorylation

- Palmitoyl CoA and AMP, in contrast, lead to the inhibition of the carboxylase.

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 Enzymes are separated Enzymes are on a single polypeptide

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 This file doesn’t contain the last chapter Amino
Acid Metabolism.

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 ‫‪THE END‬‬
‫أسأل الله أن يوفقنا جميعا لما فيه خير لنا ولأهلنا ولبلدنا وأن يملأ يومنا صلاحا وفلاحا وأن‬

‫يسخرنا لعون من نستطيع مـن الفـقراء والمكروبـين‬

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