Water

Physical Properties and Hydrogen Bonding in Water

PHYSICAL PROPERTIES OF WATER
• Water is a universal solvent
• High boiling and melting point temperatures
• High heat of vaporization
• High specific heat
• High heat of fusion
• High surface tension
• Capillary action

Hydrogen Bonding in Water

The strong intermolecular forces of attraction in liquid water are caused by the specific distribution of
electrons in the water molecule.
 Each of the 2 Hydrogen atoms share a pair of electrons with the Oxygen atom, through overlap of
the 1s orbital of the hydrogen atom with the hybridized sp3 orbital of the oxygen atom.
 From spectroscopic and x-ray analyses the precise H-O-H bond angle is 104.5 degrees , and the
average H-O inter atomic distance is 0.0965 nm.
 This arrangement of electrons in the H2O molecule give its electrical asymmetry. The highly
electronegative O atoms tend to withdraw the single electrons from the Hydrogen atoms leaving
the H-nuclei bare. As a result, each of the 2 H atoms has a local partial positive (+) charge . The
oxygen atom has a local partial negative (-) charge located in the zone of the unshared orbitals.
 Thus, although the H2O molecule has no net charge, it is an electric dipole.

H-Bonding exists only between H & O, N & F but in biomolecules the most important elements where H-
bond occurs is in O & N.
H-bonds are relatively weak bonds compared with covalent bonds. The H-bonds in liquid water are
estimated to have a bond energy of only about 4.5 k cal/mol compared with 110 k cal/mol for the covalent
H-O bonds in the water molecules.
IONIZATION OF WATER
• Water is highly polar. This suggests that water ionizes as shown below:
H2O ---------- H+ + OH-

 Hydrogen ions in aqueous solution have several names such as: hydronium ions (H3O)+, solvated
protons or hydrogen ions (H+).
The self ionization of water occurs to a very small extent. At
• 25oC, pure water has a hydrogen ion concentration [H+] equal
to its hydroxyl ion concentration [OH-]. Each Is equal to 1 X 10-7. A solution where[ H+] = [OH-]
is described as a neutral solution.
• If [ H+] > [OH-] it is an acidic solution.
• If [ H+] < [OH-] it is a basic solution.

Learning Check:
• If the [H+] = 1 x 10-5 M, is the solution acidic, basic or neutral?
• What is the [OH-] of this solution?
The pH Concept
Expressing [H+] in in moles /L or M is cumbersome. Thus the use of the pH scale was proposed by
Soren Sorensen, a Danish biochemist in 1909.

• The pH of a solution can be expressed as the negative logarithm of the [H+].

 pH = -log [H+]
In a neutral solution, the [H+] = 1 x 10-7 M. The pH of a neutral solution is 7.
In a similar way, pOH = the negative logarithm of the
[OH-].
pOH = -log [OH-]
The usual practice however, is to express both acids and bases in terms of pH.
• If the pH is known, then pOH can easily be solved since: pH + pOH = 14
• Take note:
• Neutral solution: pH = 7
• Acidic solution: pH is less than 7
• Basic solution: pH is greater than 7.
ACID-BASE THEORIES
1. Arrhenius Theory
2. Bronsted-Lowry Theory
3. Lewis Theory
The Strengths of Acids and Bases
2 classes of acids:
1. Strong acids – completely donate their protons in water because they have less affinity with their
protons

• Examples of strong acids:HCl,(hydrochloric acid) HNO3 (nitric acid), H2SO4 (sulfuric acid)

2. Weak acids – are only partially ionized in water to donate protons since they have a high affinity
with their protons. Organic acids are examples of weak acids.
The strength of a weak acid can be determined from its ionization constant, Ka.
CH3COOH ------------ CH3COO - + H+
Ka = [CH3COO-][H+]

[CH3COOH]
If the Ka of acetic acid is 1.8x10-5M at 25oC what is the [H+] of a 1 M acetic acid solution?
Let x = [H+] = [CH3COO-]
[CH3COOH] = 1M
CH3COOH -------- CH3COO- + H+
To calculate, use the Ka expression for
CH3COOH.
Ka = (x)(x)
1
1.8 x 10-5 = x2
X = 1.8 x 10-5 = 4.2 x 10-3 = 0.0042M
Thus only 0.42% of a 1M solution of acetic acid is ionized at 25 oC. Its corresponding pH value is
pH = -log[H+]
= -log(4.2x10-3)
= 2.38
Henderson-Hassellbalch equation
For a better appreciation of the role of weak acids in living systems, the equilibrium expression is most
often written in another form, called the Henderson-Hassellbalch equation. It is obtained as follows:
1. Inside the general expression HA ==== H++A-
Ka= [H+] x [A-]
[HA]
2. Rearrange the Ka expression to solve for [H+]
[H+]=Ka x [HA]
[A-]
3. Invert to logarithmic functions:
log[H+]=log Ka+log[HA]
[A-]
4. Make the expression negative
-log[H+]= -log Ka - log [HA]
[A-]
+
5. Define –log[H ] as pH and –log Ka as pKa and convert
-log[HA]/[A-] to log[A-]/[HA]

6. Henderson-Hassellbalch equation

pH=pKa + log[A-]/[HA]
= The equation is very useful in calculating the pH of the known solution and for determining the
amounts of an acid or its salt to prepare a solution of known pH.
Sample Problem: What is the pH of a solution containing 15ml of 0.1M CH3COONa and 10ml of 0.10M
CH3COOH(pKa=4.74)?
pH=4.74+log(0.10)(0.015)
(0.10)(0.010)
=4.74+log.0015
.0010
=4.74+log1.5
=4.74+0.176
=4.92
BUFFERS
Buffers are solutions in which the pH remains relatively constant when small amounts of acid or base are
added. A buffer is a solution of a weak acid and one of its salt. The acetic acid-sodium acetate
combination given in the previous section is a typical example of a buffer system. Acetic acid and its
anion, CH3COO- act as reservoirs of neutralizing power. The acetic acid contains mostly undissociated
CH3COOH which is capable of reacting with an added base by furnishing H+, thereby counteracting the
effect of added base.

CH3COOH + OH- ------- CH3COO- + H2O

The sodium acetate, on the other hand, is completely ionized. When an acid is added to the solution the
CH3COO- act as a “hydrogen ion sponge”. This creates CH3COOH which does not dissociate extensively
in water. Thus the pH does not change appreciably.

CH3COO- +H+-----CH3COOH

The pH of a buffer solution after the addition of acid(a) and base(b) may be computed from the following
equations.
pH=pKa +log[salt-a]
[acid+a]

pH=pKa +log[salt+b]
[acid-b]

-An acetate buffer cannot control the pH when too much acid is added. Then no more acetate ions are
present to accept H+. The buffer also becomes ineffective when too much base is added.
-Then no more acetic acid molecules are present to donate H+ to neutralize the OH-.
The buffer capacity is the amount of acid or base that may be added to a buffer solution before a
significant change in pH occurs.
Control of pH in Body Fluids
Living systems are intricate chemical factories. They are desirable but delicate systems. A myriad of
chemical reactions are constantly taking place within them. Most of these reactions produce organic acids
which add protons as H+ to the system which in turn will affect the pH of the system. However, body pH
should be maintained at a constant value. The importance of pH control is illustrated in the case of human
blood, whose pH must be kept within narrow limits for life and even narrower limits for health, as
illustrated below.
Blood plasma has a normal pH of 7.4. If the pH should fall below 7.0 or rise above 7.8 the results would
be fatal. Physiological pH is efficiently maintained by a buffer system. The major buffer system in the
blood is the carbonic acid-bicarbonate system which is very effective in protecting this fluid from large
changes in pH.

How does this buffer system protect the blood? Consider the following equilibrium equation:

H2CO3--------HCO3- + H+
Addition of a strong acid to blood will increase the H+ and will consequently drive the reaction to the left
forming more carbonic acid. But carbonic acid is unstable and will decompose to form carbon dioxide
and water.

H2CO3-------CO2(g) + H2O

The carbon dioxide formed is removed from the blood

and exhaled through the lungs. The buffer is also
effective when a strong base is added to blood. The
mechanisms against both acidosis and alkalosis are
illustrated in the figure below.

Acidosis
CO2 + H2O -------H2CO3 -----HCO3- + H+ ---- H+

CO2 is rapidly exhaled; HCO3- is retained in blood; H+

is excreted in urine

Alkalosis
CO2 +H2O ----- H2CO3 -----H+ +HCO3- ----HCO3-

CO2 + H2O are retained by slow breathing;

H+ is retained in blood; HCO3- is excreted in urine
Summary

Water is the most abundant compound in living organisms. Its unique properties make it most suitable as
the solvent of life. Mainly responsible for these properties are its polarity and H-bonding capacity.
Acids are defined as proton donors and bases as proton acceptors. An acid/conjugate base pair refers to a
proton donor and its corresponding proton acceptor. Weak acids which display a strong affinity for their
protons are of great biological importance since they act as buffers to resist changes in pH and therefore
help to maintain normal internal pH in organisms.
In humans the main extracellular buffering system is the bicarbonate buffer (H 2CO3/HCO3- pair) and the
principal intracellular system is the phosphate buffer, specifically that which involves the second
dissociation constant,Ka2 (H2PO4-/HPO42- pair).
References:
Biochemistry by: Campbell
Lehninger’s Principles of Biochemistry by; Cox, et al.
On-Line references
 Amino Acids and Peptides

• Amino acid: a compound that contains both an

amino group and a carboxyl group
• a-Amino acid has an amino group attached
to the carbon adjacent to the carboxyl group
• -carbon also bound to side chain group, R
• R gives identity to amino acid
• Two steroisomers of amino acids are
designated L- or D-. Based on similarity to
glyceraldehdye (Figure 3.2)

Amino Acid Structure and Properties

• With the exception of glycine, all protein-derived amino acids have at least one
stereocenter (the a-carbon) and are chiral (stereoisomers)
• the vast majority of a-amino acids have the L-configuration at the a-carbon
(Proline is usually D)
• Side-chain carbons in other amino acids designated with Greek symbols, starting at a
carbon (…etc)
• Amino acids can be referred to by three-letter or one-letter codes. Table 3.
Individual Amino Acids
Group A: Nonpolar side chains- Ala, Val, Leu, Ile, Pro. Phe, Trp, Met.
• Ala, Val, Leu, Ile, Pro- contain aliphatic hydrocarbon group. Pro has cyclic structure.
• Phe- hydrocarbon aromatic ring.
• Trp- Indole ring side chain, aromatic.
• Met- Sulfur atom in side chain.
Amino Acids (cont’d)
 Group B: Neutral Polar side chains- Ser, Thr, Tyr, Cys, Glu, Asn
• Ser, Thr- Side chain is polar hydroxyl group
• Tyr- hydroxyl group bonded to aromatic hydrocarbon group
• Cys- Side chain contains thiol group (-SH)
• Gln, Asn- contain amide bonds in side chain
• Group C: Acidic Side Chains: Glu, Asp
• Both have a carboxyl group in side chain
• Can lose a proton, forming a carboxylate ion
• These amino acids are negatively charged at neutral pH
• Group D: Basic side chains: His, Lys, Arg
• Side chains are positively charged at pH 7
• Arg-side chain is a guanidino group
• His-side chain is an imidazole group
• Lys-side chain NH3 group is attached to an aliphatic hydrocarbon chain

Amino acid summary

Important structural features:
1. All 20 are a-amino acids
2. 19 of the 20, the a-amino group are primary; for proline, it is secondary
3. With the exception of glycine, the a-carbon of each is with a stereocenter
4. Isoleucine and threonine contain a second stereocenter
5. 3, and 1-letter codes in Table 3.1.
Uncommon Amino Acids

• Each derived from a common amino

acid by a modification
• hydroxylysine and
hydroxyproline are found
only in a few connective
tissues such as collagen
• thyroxine is found only in
the thyroid gland

Ionization of Amino Acids

-In amino acid, carboxyl group (-) and
amino group (+) are charged at neutral
pH.
-In free amino acids -carboxyl, and a-amino
groups have titratable protons. Some side
chains do as well
- Remember, amino acids without charged

groups on side chain exist in neutral solution as zwitterions with no net charge
Titration of Amino Acids
• When an amino acid is titrated, the
titration curve represents the reaction of each
functional group with the hydroxide ion

Titration of alanine with NaOH

Titration of histidine with NaOH

Acidity: a-COOH Groups

 • The average pK of an a-carboxyl group is 2.19, which makes them considerably
a
stronger acids than acetic acid (pK 4.76)
a
• the greater acidity of the amino acid carboxyl group is due to the electron-withdrawing
+
inductive effect of the -NH group
3
+
Basicity: a-NH3 groups
• The average value of pKa for an a-NH3+ group is 9.47, compared with a value of 10.76 for a 2°
Basicity (cont’d) ion
alkylammonium
Guanidine Group
• The side chain of arginine is a considerably stronger base than an aliphatic amine
• basicity of the guanido group is attributed to the large resonance stabilization of
pKa = 2.00
-
COOH + H2 O COO + H3 O +
the protonated form relative to the neutral form
Imidazole Group
• The side chain imidazole group of histidine is a
heterocyclic aromatic amine

Ionization vs pH
• Given the value of pKa of each functional group, we can calculate the ratio of each acid to
its conjugate base as a function of pH
• Consider the ionization of an a-COOH

• writing the acid ionization constant and rearranging terms give (remember Ch. 2)

[ H 3 O + ] [ -COO - ] [ -COO - ] Ka
Ka = or =
[ -COO H] [ -COO H] [ H3 O+ ]

Ionization vs pH (cont’d)
 • substituting the value of Ka (1 x 10-2) for the hydrogen ion concentration at pH 7.0 (1.0 x
10-7) gives
[ -NH 2 ] Ka
=
[ -NH 3
+
] [H 3 O+ ] • at pH 7.0, the a-
carboxyl group is virtually 100%
in the ionized or conjugate base form, and has a net charge of -1
• we can repeat this calculation at any pH and determine the ratio of [a-COO -] to [a-
COOH] and the net charge on the a-carboxyl at that pH

• We can also calculate the ratio of acid to conjugate base for an a-NH3+ group; for this
calculation, assume a value 10.0 for pKa
• writing the acid ionization constant and rearranging gives

Ionization vs Ph
• substituting
values for Ka of
an a-NH3+ group
and the hydrogen ion concentration at pH 7.0 gives
• at pH 7.0, the ratio of a-NH2 to a-NH3 + is approximately 1 to 1000
• at this pH, an a-amino group is 99.9% in the acid or protonated form and has a charge of
+1

Henderson-Hasselbalch Equation
• We have calculated the ratio of acid to conjugate base for an a-carboxyl group and an a-
amino group at pH 7.0
• We can do this for any weak acid and its conjugate base at any pH using the Henderson-
Hasselbalch equation.
 Isoelectric pH
• Isoelectric pH, pI: the pH at which the majority of
molecules of a compound in solution have no net charge

• the pI for glycine, for example, falls midway

between the pKa values for the carboxyl and amino
groups

• Isoelectric pH values for the 20 protein-derived amino acids are given in Table 3.2

Electrophoresis

• Electrophoresis: the process of separating compounds on the basis of their electric charge

Peptide Bonds
• electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose
acetate as solid supports
• Individual amino acids can be linked by forming covalent bonds.
• in paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as
• aPeptide bond: the special name given to the amide bond between the a-carboxyl group
bridge between two electrode vessels
of one amino acid and the a-amino group of another amino acid
Geometry of Peptide Bond
• the four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles
of 120° about C and N

• to account for this geometry, a peptide bond is

most accurately represented as a hybrid of two
contributing structures (resonance structures)
• the hybrid has considerable C-N double
bond character and rotation about the peptide bond is restricted
Peptides
peptide: the name given to a short
polymer of amino acids joined by peptide bonds;
they are classified by the number of amino acids in the chain
• dipeptide: a molecule containing two amino acids joined by a peptide bond
• tripeptide: a molecule containing three amino acids joined by peptide bonds
• polypeptide: a macromolecule containing many amino acids joined by peptide bonds
• protein: a biological macromolecule of molecular weight 5000 g/mol or greater, consisting of
one or more polypeptide chains
Peptides with Physiological Activity Peptides with Physiological Activity (cont’d)

Protein Structure
• Many conformations are possible for proteins:
• Due to flexibility of amino acids linked by peptide bonds
• At least one major conformation has biological activity, and hence is considered the protein’s
native conformation
Levels of Protein Structure
1° structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end
to the C-terminal end
2° structure: the ordered 3-dimensional arrangements
(conformations) in localized regions of a polypeptide
chain; refers only to interactions of the peptide backbone
• e. g., a-helix and b-pleated sheet
3˚ structure: 3-D arrangement of all atoms
4˚ structure: arrangement of monomer subunits with
respect to each other

1˚ Structure
• The 1˚ sequence of proteins determines its 3-D conformation
 • Changes in just one amino acid in sequence can alter biological function, e.g. hemoglobin
associated with sickle-cell anemia
• Determination of 1˚ sequence is routine biochemistry lab work (See Ch. 5).
2˚ Structure
• 2˚ structure of proteins is hydrogen-bonded arrangement of backbone of the protein
• Two bonds have free rotation:
1) Bond between -carbon and
amino nitrogen in

residue
2) Bond between the -carbon and carboxyl carbon of residue
• See Figure 4.1

a-Helix
• Coil of the helix is clockwise or right-handed
• There are 3.6 amino acids per turn
• Repeat distance is 5.4Å (5.4 Angstrom)
• Each peptide bond is s-trans and planar
• C=O of each peptide bond is hydrogen bonded to the N-H of the fourth amino acid away
• C=O----H-N hydrogen bonds are parallel to the helical axis
• All R groups point outward from helix

a-Helix (cont’d)
 •
•
•
•

• Several factors can disrupt an a-

helix
• proline creates a bend because
of (1) the restricted rotation
due to its cyclic structure and
(2) its a-amino group has no
N-H for hydrogen bonding
• strong electrostatic repulsion caused by the proximity of several side chains of like
charge, e.g., Lys and Arg or Glu and Asp
• steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr
b-Pleated Sheet
• Polypeptide chains lie adjacent to one another; may be parallel or antiparallel
• R groups alternate, first above and then below plane
• Each peptide bond is s-trans and planar
• C=O and N-H groups of each peptide bond are perpendicular to axis of the sheet
• C=O---H-N hydrogen bonds are between adjacent sheets and perpendicular to the
direction of the sheet
b-Pleated Sheet (Cont’d)
-bulge- a common nonrepetive irregular 2˚ motif in anti-parallel structure
Structures of Reverse Turns
Glycine found in reverse turns
• Spatial (steric) reasons
• Polypeptide changes direction
• Proline also encountered in reverse turns. Why
a-Helices and b-Sheets
• Supersecondary structures: the combination of a- and b-sections, as for example
• bab unit: two parallel strands of b-sheet connected by a stretch of a-helix
• aa unit: two antiparallel a-helices
• b-meander: an antiparallel sheet formed by a series of tight reverse turns
connecting stretches of a polypeptide chain
• Greek key: a repetitive supersecondary structure formed when an antiparallel sheet
doubles back on itself
• b-barrel: created when b-sheets are extensive enough to fold back on themselves
Schematic Diagrams of Supersecondary Structures
Collagen Triple Helix

• Consists of three polypeptide chains

wrapped around each other in a ropelike
twist to form a triple helix called
tropocollagen; MW approx. 300,000
• 30% of amino acids in each chain are Pro
and Hyp (hydroxyproline); hydroxylysine
also occurs
• Every third position is Gly and repeating
sequences are X-Pro-Gly and X-Hyp-Gly
• Each polypeptide chain is a helix but not
an a-helix
• The three strands are held together by
hydrogen bonding involving
hydroxyproline and hydroxylysine
Fibrous Proteins
• With age, collagen helices become cross
• Fibrouslinked by covalent
proteins: containbonds formed between
polypeptide
Lys and Hisapproximately
chains organized residues parallel
along a single axis. They
• consist of long fibers or large
sheets
• tend to be mechanically strong
• are insoluble in water and dilute
salt solutions
• play important structural roles in
nature
• Examples are
• keratin of hair and wool
 •collagen of connective tissues of animals including cartilage, bones, teeth, skin,
and blood vessels
Globular Proteins
• Globular proteins: proteins which are folded to a more or less spherical shape
• they tend to be soluble in water and salt solutions
• most of their polar side chains are on the outside and interact with the aqueous
environment by hydrogen bonding and ion-dipole interactions
• most of their nonpolar side chains are buried inside
• nearly all have substantial sections of a-helix and b-sheet
Comparison of Shapes of Fibrous and
Globular Proteins

3˚ Structure
• The 3-dimensional
arrangement of
atoms in the molecule.
• In fibrous protein, backbone of protein does not fall back on itself, it is an important
aspect of 3˚ not specified by 2˚ structure.
• In globular protein, more information is needed. 3k structure allows for the
determination of the way helical and pleated-sheet sections fold back on each other.
Interactions between side chains also play a role
Forces in 3˚ Structure
• Noncovalent interactions, including
• hydrogen bonding between polar side chains, e.g., Ser and Thr
• hydrophobic interaction between nonpolar side chains, e.g., Val and Ile
• electrostatic attraction between side chains of opposite charge, e.g., Lys and Glu
• electrostatic repulsion between side chains of like charge, e.g., Lys and Arg, Glu
and Asp
• Covalent interactions: Disulfide (-S-S-) bonds between side chains of cysteines

Forces That Stabilize Protein Structure

3° and
4°

Structure
• Tertiary (3°) structure: the arrangement in space of all atoms in a polypeptide chain
• it is not always possible to draw a clear distinction between 2° and 3° structure
• Quaternary (4°) structure: the association of polypeptide chains into
aggregations
• Proteins are divided into two large classes based on their three-dimensional structure
• fibrous proteins
• globular proteins

Determination of 3° Structure
 • X-ray crystallography
• uses a perfect crystal; that is, one in which all individual protein molecules have
the same 3D structure and orientation
• exposure to a beam of x-rays gives a series of diffraction patterns
• information on molecular coordinates is extracted by a mathematical analysis
called a Fourier series
• 2-D Nuclear magnetic resonance
• can be done on protein samples in aqueous solution
X-Ray and NMR Data

Determines solution structure

High resolution method to determine 3˚
structure of proteins (from crystal) Structural info. Gained from determining distances
between nuclei that aid in structure determination
Diffraction pattern produced by electrons
scattering X-rays

Series of patterns taken at different

angles gives structural information

Myoglobin

• A single polypeptide chain of 153 amino acids

• A single heme group in a hydrophobic pocket
• 8 regions of a-helix; no regions of b-sheet
• Most polar side chains are on the surface
• The Structure
Nonpolar of Myoglobin
side chains are folded to the interior Oxygen Binding Site of Myoglobin
• Two His side chains are in the interior, involved with interaction with the heme group
• Fe(II) of heme has 6 coordinate sites; 4 interact with N atoms of heme, 1 with N of a His side chain, and 1 with
either an O2 molecule or an N of the second His side chain
Denaturation
• Denaturation: the loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a protein
its biological activity; that is, the loss of biological activity
• Denaturation can be brought about by
• heat
• large changes in pH, which alter charges on side chains, e.g., -COO- to -COOH or -NH3+ to -NH2
• detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions
• urea or guanidine, which disrupts hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds

Denaturation of a Protein
Denaturation and
Refolding in Ribonuclease
Several ways to denature
proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride
Quaternary (4°) structure:
• Quaternary (4°) structure: the association of polypepetide monomers into multisubunit
proteins
• dimers
• trimers
• tetramers
• Noncovalent interactions
• electrostatics, hydrogen bonds, hydrophobic

Oxygen Binding of Hemoglobin (Hb)

• A tetramer of two a-chains (141 amino acids each) and two b-chains (153 amino acids
each); a2b2
• Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2
• Binding of O2 exhibited by positive cooperativity; when one O2 is bound, it becomes
easier for the next O2 to bind
• The function of hemoglobin is to transport oxygen
• The structure of oxygenated Hb is different from that of unoxygenated Hb
• H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and
transport oxygen
Structure of Hemoglobin Conformation Changes That Accompany Hb Function
Structural changes occur during binding of small
molecules
• Characteristic of allosteric behavior
• Hb exhibits different 4˚ structure in the bound and
unbound oxygenated forms
• Other ligands are involved in cooperative effect of Hb
can affect protein’s affinity for O2 by altering structure

Oxy- and Deoxyhemoglobin

 Protein Folding Dynamics
Can 3˚ structure of protein be predicted? Yes, within limitations
• The integration of biochemistry and computing has led to bioinformatics
• Protein structure prediction is one of the principal application of bioinformatics
• First step to predict protein structure is to search for sequence homology

Predicting Protein Structure

Hydrophobic Interactions

Hydrophobic interactions are major factors in protein folding

• Folds so that nonpolar hydrophobic side chains tend to be on inside
away from water, and polar side chains on outside accessible to
aqueous environment
• Hydrophobic interactions are spontaneous

Hydrophobic and Hydrophilic Interactions in Proteins

AMINO ACID SEQUENCING

Protein Folding Chaperones
N-TERMINUS
• In the protein-dense environment of a cell,
- Edman Degradation: uses phenylisothiocyanate
proteins may begin to fold incorrectly or may
(PITC); cycle repeated
associate with other proteins before folding is
C-TERMINUS : uses exopeptidases (carboxypeptidase), and
completed
Hydrazine
• Special proteins called chaperones aid in the
INTERNAL RESIDUES:
correct and timely folding of many proteins
- Trypsin which cleaves –CONH donated by basic
• hsp70 were the first chaperone proteins
amino acids;
discovered
- Chymotrypsin which cleaves –CONH donated by
• Chaperones exist in organisms from
ENZYMES aromatic amino acids;
prokaryotes to humans
- Cyanogen Bromide which cleaves –CONH donated
by methionine
  Enzymes are inseparable from living organisms. They are the largest group of proteins
and they catalyze almost every reaction that occurs in living organisms. They exhibit
extraordinary catalytic powers,
 much greater than that of synthetic catalysts. They exhibit a high degree of specificity &
they function in dilute aqueous solutions under very mild conditions of temperature and
pressure
 An enzyme is an organic compound that acts as a catalyst for a biochemical reaction.
 They are the largest and the most highly specialized class of proteins.

Enzyme structure
 Enzymes are proteins Human pancreatic amylase
 They have a globular shape
 A complex 3-D structure
It has two general structural classes
 Simple enzymes
 Conjugated enzymes

 Simple enzymes
are enzymes composed only of protein (amino acid chains)
 Conjugated enzymes
are enzymes that have a non-protein part in addition to their protein part
Special Terms Used in Enzymology:
 For complex enzymes:
 Apoenzyme is the protein part of the enzyme.
Co-enzyme or prosthetic group is the non protein part. Together, the apoenzyme and the
coenzyme make up the holoenzyme
Structure of Enzyme
 Coenzyme is a small organic molecule that serves as a cofactor in a conjugated enzyme
 Nitrogenase enzyme with Fe, Mo and ADP cofactors

Cofactors
 An additional non-protein molecule that is needed by
some enzymes to help the reaction
 Tightly bound cofactors are called prosthetic groups
 Cofactors that are bound and released easily are called
coenzymes
 Nitrogenase enzyme with Fe, Mo and ADP cofactors
 Many vitamins are coenzymes
Special Terms Used in Enzymology
 For complex enzymes:
 Apoenzyme is the protein part of the enzyme.
 Co-enzyme or prosthetic group is the non
protein part. Together, the apoenzyme and the coenzyme make up the holoenzyme.

Nomenclature and Classification of Enzymes

 Enzymes are most commonly named by using a system that attempts to provide
information about the function rather than the structure of the enzyme
 Substrate is the reactant in an enzyme-catalyzed reaction

Important aspects of enzyme-naming process

 The suffix –ase identifies a substance as an enzyme (urease, sucrase & lipase)
 The suffix –in is still found in the names of some of the first enzymes studied, many of
which are digestive enzymes (trypsin,chymotrypsin & pepsin)
 The type of reaction catalyzed by an enzyme is often noted with a prefix (oxidase
&hydrolase)
 The suffix –ase identifies a substance as an enzyme (urease, sucrase & lipase)
 The suffix –in is still found in the names of some of the first enzymes studied, many of
which are digestive enzymes (trypsin,chymotrypsin & pepsin)
 The type of reaction catalyzed by an enzyme is often noted with a prefix (oxidase &hydrolase)
Predict the function of the following enzyme
 Cellulase
 cellulase catalyzes hydrolysis of cellulose
 Sucrase
 sucrase catalyzes hydrolysis of the disaccharide sucrose
 L-amino acid oxidase
 catlayzes the oxidation of L-amino acids
 Aspartate aminotransferase
 aspartate aminotransferase catalyzes the transfer of an amino group from aspartate to a
different molecule of L-amino acids
 Six major classes of enzymes on the basis of the types of reactions they catalyse
 An oxidoreductase
an enzyme that catalyzes an oxidation-reduction reaction

 A transferase
an enzyme that catalyzes the transfer of a functional group from one molecule to another
It has two major subtypes
1. transaminases
2. kinases
 Transaminases transferase that catalyzes the transfer of an amino group from one
molecule to another
 Kinases transferase that play a major role in metabolic energy-production reaction,
catalyze the transfer of a phosphate group from ATP to ADP and a phosphorylated
product
 Kinases transferase that plays a major role in metabolic energy-production. The reaction
catalyzes the transfer of a phosphate group from ATP to ADP and a phosphorylated
product

 Lyase is an enzyme that catalyzes the addition of a group to a double bond or the
removal of a group to form a double bond in a manner that does not involve hyrdrolysis
or oxidation (ex. dehydratase & hydratase)
 hydrolase is an enzyme that catalyzes a hydrolysis reaction in which the addition of
water molecule to a bond causes the bond to break

 Isomerase is an enzyme that catalyzes the isomerization (rearrangement of atoms) of a

substrate in a reaction, into a molecule isomeric with itself there is only one reactant and
product in reactions where isomerases are operative

 Ligase is an enzyme that catalyzes the bonding together of two molecules into one with
the participation of ATP
-ATP is required because such reactions are generally energetically unfavorable and they
require the simultaneous input of energy obtained by hydrolysis reaction in which ATP is

converted to ADP
 MODEL OF ENZYME ACTION
The active site

 One part of an enzyme, the active site, is

particularly important
 The shape and the chemical environment
inside the active site permit a chemical
reaction to proceed more easily
 A small portion of an enzyme molecule that
pariticipates in the interaction with a
substrate/s during the reaction
 It is actually the part of the enzyme that is
involved in the catalysis

The substrate
 The substrate of an enzyme is the reactant that is activated by the enzyme
 Enzymes are specific to their substrates
 The specificity is determined by the active site
The Lock and Key Hypothesis
 Fit between the substrate and the active site of the enzyme is exact
 Like a key fits into a lock very precisely
 The key is analogous to the enzyme and the substrate analogous to the lock.
 Temporary structure called the enzyme-substrate complex formed
 Products have a different shape from the substrate
 Once formed, they are released from the active site
 Leaving it free to become attached to another substrate
S E
E
E

Enzyme- Enzyme may

substrate be used again
complex P

P
The Lock and Key Hypothesis
 This explains enzyme specificity
 This explains the loss of activity when enzymes denature

The Induced Fit Hypothesis

 Some proteins can change their shape (conformation)
 When a substrate combines with an enzyme, it induces a change in the enzyme’s
conformation
 The active site is then moulded into a precise conformation
 Making the chemical environment suitable for the reaction
 The bonds of the substrate are stretched
to make the reaction easier (lowers
activation energy)

 This explains the enzymes that can react

with a range of substrates of similar types

Factors affecting Enzymes

 substrate concentration
 pH
 temperature
 inhibitors
Substrate concentration: Non-enzymic
reactions
 The increase in velocity is
proportional to the substrate
concentration

Substrate concentration: Enzymic reactions

 Faster reaction but it reaches a saturation
point when all the enzyme molecules are
occupied.

 If you alter the concentration of the enzyme

then Vmax will change too.
  Extreme pH levels will produce denaturation
The effect of pH
 The structure of the enzyme is changed
 The active site is distorted and the substrate
molecules will no longer fit in it
 At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of
the enzyme and it’s substrate molecules will occur
 This change in ionization will affect the binding of
the substrate with the active site

The effect of temperature

 Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in
temperature.
 For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every 10°C rise in temperature)

 Enzyme-controlled reactions follow this rule as they are chemical reactions

 But at high temperatures proteins denature
 The optimum temperature for an enzyme controlled reaction will be a balance
between the Q10 and denaturation.
 For most enzymes the optimum temperature is about 30°C
 Many are a lot lower,
cold water fish will die at 30°C because their enzymes denature
 A few bacteria have enzymes that
can withstand very high
temperatures up to 100°C
 Most enzymes however are fully
denatured at 70°C
 E+I EI

Reversible Enzyme inhibitor

reaction complex

Inhibitors
 Inhibitors are chemicals that reduce the rate of enzymic reactions.
 They are usually specific and they work at low concentrations.
 They block the enzyme but they do not usually destroy it.
 Many drugs and poisons are inhibitors of enzymes in the nervous system.
The effect of enzyme inhibition
 Irreversible inhibitors: Combine with the functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus,
combine with serine residues in the
enzyme acetylcholine esterase.
 Reversible inhibitors: These can be

washed out of the solution of enzyme by

dialysis.
There are two categories:
1. Competitive: These compete with the
substrate molecules for the active site.
The inhibitor’s action is proportional to its
concentration.
Resembles the substrate’s structure closely.

2. Non-competitive: These are not influenced by the concentration of the substrate. It

inhibits by binding irreversibly to the enzyme but not at the active site.
Examples
 Cyanide combines with the Iron in the enzymes cytochrome oxidase.
 Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a
chelating agent such as EDTA.

Allosteric Regulation
 Allosteric regulation is a form of non-competitive inhibition. It involves an oligomeric
enzyme (more than one polypeptide protein) which contains sites at which substrate
molecules can bind (active site) & sites at which inhibitor molecules can bind (allosteric
 site). There is an interaction between the substrate and the inhibitor such that when an
inhibitor occupies an allosteric
 site the active site changes conformation. If the change effects an increase in the rate of
the reaction, the inhibitor also called allosteric effector is properly referred to as positive
effector or allosteric activator.
 It is called negative effector or allosteric inhibitor if reaction rate is decreased.
Applications of inhibitors
 Negative feedback: end point or end product inhibition
 Poisons snake bite, plant alkaloids and nerve gases.
 Medicine antibiotics,
sulphonamides, sedatives and
stimulants

Chemical reactions
 Chemical reactions need an initial
input of energy = THE ACTIVATION
ENERGY
 During this part of the reaction the
molecules are said to be in a transition
state.

Reaction pathway
Making reactions go faster

 Increasing the temperature makes

molecules move faster
 Biological systems are very sensitive to
temperature changes.
 Enzymes can increase the rate of reactions
without increasing the temperature.
 They do this by lowering the activation
energy.
 They create a new reaction pathway “a
short cut”

An enzyme controlled pathway

 Enzyme controlled reactions
proceed 108 to 1011 times faster
than corresponding non-
enzymatic reactions.