Lecture Study Guide Bioc100C Spring 2015

What is Biochemistry? - Addresses following questions:

What are the molecules in living organisms?
How are they made?
How are they recycled?
Where does the energy come from to do this chemistry?
Metabolism is about food very relevant
This course will provide answers to many practical questions
1. Which will produce more ATPs, more calories?
1 oz of butter, bread, chicken breast?
What is the molecular composition of butter, bread, chicken ?
2. Can you make:
fat from sugar?
Sugar from fat?
Amino acids from sugar?
Sugar from amino acids?
3. What are vitamins?
Name a vitamin and give its biochemical function
What do you need to grow cells?
simple cells - some photosynthetic bacteria
air (CO2, N2, O2), Pi, SO4, K Na, Cl, Mg Fe, trace minerals,
Mammalian cells,
10 amino acids
12 vitamins
choline
inositol
polyunsaturated fatty acids (the bad kind)
minerals (at least 13)
thus only about 25 organic molecules, organism makes rest
How many kinds of molecules in a cell?
E. coli a well-studied bacterium
4,400 genes (1 molecule of DNA)
3000 RNAs (polycistronic)
3000 proteins
500-1000 small molecules, ATP, amino acids, steroids etc
thus about 7000 different kinds (counting the proteins and RNAs)
Mammalian cells 1000X more DNA but only 6X more genes
Maybe 50,000 different molecules? (most of variety is in RNA and proteins.)
How are these made and degraded?
Metabolism - conversion of one molecule into another
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Anabolic - Biosynthesis, putting molecules together

Catabolic - Degradative, taking them apart
How to think about these molecules
Size - molecular mass (kDa)
dimensions in (atom), nm small molecules, micron (bacterium)
Composition -with Nitrogen?, other molecules?
Structure - rings? globular?, linear?, monomer? polymer?
Function
Free energy, ATP
A. the reactions in cells have to conform to thermodynamic principles
B. The energy to make/degrade molecules is often provided by ATP or by redox
reactions
First - Biology has to obey the laws of physics
1st law - the total amount of energy remains constant
2nd law in any process of the entropy of the universe will increase
(3rd - the entropy of a crystal is 0 at 0 Kelvin)
Primary rule that reactions must follow is:
G = H -T S
G = free energy (biochemical useable energy)
H = change in energy in chemical bonds
T = temperature in K
S = change in entropy - disorder or randomness
for a reaction to proceed, G must be negative, must yield free energy
We can determine the free energy from the equilibrium constant
keq = products /reactions
G = -RTlnKeq

R = 8.3 J/mol.deg

R and T are always positive, therefore G will be positive or negative

depending on Keq
If at equilibrium, there are more products than reactants G is negative
Two important points about the use of G
1. G for a reaction in a cell may be significantly different then the "standard G "
because, reaction will not be at equilibrium, concentration of substrates and products
will be different
this effect called "mass action"
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2. G can be use to analyze a whole series of reactions

If A B C
A B G = 10 kJ
B C G = -30 kJ
A C

G = -20 kJ

The point is that individual steps in a pathway may have positive Gs

but will still go forward, often because of "mass action"
Importance of diffusion - This is what limits the maximum rate of a reaction
In a cell this number is ~ 1 million per mmol per sec
(I wont test you on section 13.2, chemical reaction mechanisms)
Why is ATP a "high energy " molecule?
Hydrolysis of gamma Pi yields -30 kJ/mol
electrons going from higher to lower orbitals
a. relieve charge repulsion between oxygens
b. resonance stabilization of charge on Pi
c. proton dissociates (e.g. ADP product splits into two molecules, increases entropy)
Other phophorylated compounds are used as high energy intermediates
GTP, UTP, PEP, biphosphogylcerate, glucose-phosphate
Pyrophosphate (PPi), used in plants, not in other organisms
Polyphosphate,
Worth remembering relative energy from these molecules
Fig 13.9, PEP > Bis-P glucose > P-creatine > ATP > Glucose-P
(You can make ATP with first 3)
Hydrolysis, per se, is not the usual way to use the free energy
typically group transfer
phosphoryl group covalently bound to enzyme or substrate intermediate,
sometimes, adenylate is bound
ATP rarely is naked ATP-4, almost always bound to Mg+2
ATP is very stable in cells
Oxidation-reduction reactions
Many important biochemical reactions are electron transfer reactions
Cells get energy by oxidizing our food. We combine sugars, fats, proteins, with oxygen.
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(we burn our food, slowly, produce CO2 and H2O)

In cells oxidation often involves oxygen, but not always.
Oxidation and reduction refer to the transfer of electrons
Example Fe+2 + Cu+2 -> Fe+3 + Cu+ (ferrous -> ferric, cupric -> cuprous)
Ferrous ion gets oxidized - an electron is removed
Cupric ion get reduced, it gains an electron (gets more minus)
Oxidation state of an atom defines how many electrons it owns
(i.e., how many electrons spend most of their time around its nucleus)
Fig 13-22 illustrates amount of energy to be gotten by oxidizing each electron
Reduction Potential what does this term mean?
A measure of the affinity of a molecule for an electron
Diagram 13-23, explain how reduction potentials are measured
Examples of reduction potentials
H+ + e- -> H2

Eo' is 0

O2 + 2 e- + 2H+ H2O

Eo' is + 0.816 volts

NAD+ + H+ + 2e- NADH

- 0.324 volts

cyto c Fe+3 + e- Fe+2

"

+ 0.29 volts

oxygen has a greater affinity for electrons than cyto c and thus the
electrons from cyto c can be passed on to oxygen if appropriate enzymes
are present.
some are good donors - e.g. NADH a reducing agent
some are good acceptors e.g. oxygen, an oxidizing agent
What is the relationship between G and E?
Look at worked example 13-3
What are the units? G is in Joules per mol (or calories per mol)
E is in Volts
G = -nF E
n = number of electrons transferred in the reaction
F = 96.5 kJ/V.mol (Faraday constant)
E = difference in reduction potential, in volts
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Thus if one electron is transferred, and the E = 0.5 Volt, then

G = -1 X 96.5 kJ/Volt.mol X 0.5 Volt
G = -48.2 kJ/mol
These equivalencies are useful:
10/1 concentration gradient = 60 mVolts = 5.8 kJ/mol
Several molecules are specialized electron carriers that function in many reactions.
NADH and NADPH carry high energy electrons
soluble molecules, but often bind enzymes during electron transfer
NADH usually in catabolic pathways, harvesting energy
NADPH usually in anabolic pathways, donates to makes molecules that
contain high energy electrons
Niacin (= nicotinic acid) used to make NADH
Deficiency in dietary niacin can cause Pellagra, we can make some niacin from
trptophan.
Flavins also carry electrons
not soluble, cofactors in enzymes
FMN - Flavin mononucleotide
FAD - Flavin adenine dinucleotide (contains an FMN)
bind 2 e- and 2 H+
generally bind e- of lower energy than NAD
affinity affected by the protein they are in.
Glycolysis
perhaps the most important pathway in the cell
present in essentially all organisms
makes intermediates used in most other pathways
good example of a metabolic pathway
means sugar lysis
glucose 2 pyruvates, make 2 ATPs, 2 NADHs
this is done in 10 steps
glucose + 2 ATP 2 glyceraldehyde-P
2 glyceraldehyde-P + 4 ADP + 2 NAD+ 2 pyruvates + 2 NADH, + 4
ATP
Look at the pathway in detail (Put on board, transparency on overhead)
1. Glucose + ATP G-6-P
essentially gives activated glucose, more reactive
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hexokinase (kinases, puts Pi on hexoses)

G = -16.7 kJ
2. G-6-P F-6-P
rearranges the molecule
P-glucose isomerase
G = +1.7 kJ (essentially 1:1 at equilibrium)
3. F-6-P + ATP F-1,6,bisphosphate
fully activated 6C sugar
phosphofructokinase (note meaning of name)
G = -14.2 kJ
Note that plants use PPi, more efficient is some ways
committed step in glycolysis
F-6-P can be used elsewhere
highly regulated enzyme
4. F-1,6,bis-P DHA-P + G-3-P
splits into 2 reactive 3C-P
aldolase (reverse is an aldol condensation)
G = +23.8 kJ note large +value
5. DHA-P G-3-P
isomerize, get two G-3-P per glucose
triose phosphate isomerase
G = +7.5 kJ
6. G-3-P + Pi + NAD+ 1,3-bis-P + NADH
harvest a pair of electrons and add another P-group
G-3-P dehydrogenase
G = +6.3 kJ
example of an acyl-P, "high energy" (G = -49 kJ)
probably most complex reaction in the pathway
good one to look at mechanism
Overhead
a. enzyme has NAD+ and cys-SH at active site
b. bind G-3-P to -S
c. pass H- (hydride ion) to NAD+
d. NADH passes H- to soluble NAD+
e. phosphorolysis of S-C bond
f. release 1,3-bis-G
7. 1,3-P-G + ADP 3-P-G + ATP
make ATP by group transfer
P-glycerate kinase
G = -18 kJ
(remember, acyl-P -49kJ, ATP, -30 kJ)
our first example of "substrate-level phosphorylation"
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8. 3-P-G 2-P-G
isomerization
P-glycerate mutase
G = +4.4 kJ
interesting mechanism
a. 3-P-G + His-P
b. 2,3-P-G + His
c. 2-P-G + His-P
9. 2-P-G PEP + H2O
remove H2O to generate high energy phosphate group
enolase (make enol C=C)
G = + 7.5
redistribute energy, change phosphate bond
10. PEP + ADP pyruvate + ATP
make ATP by group transfer
pyruvate kinase
G = - 31kJ
Note large free energy even though ATP is made
energy of hydrolysis of PEP is -61 kJ
makes this reaction irreversible
"pulls" whole pathway
also, non-enzymatic step, enol pyruvate keto pyruvate
What do you need to know? You should be able to show the following:
Know the summary reaction:
Glucose + 2 ATP + 2 NAD+ -> 2 Pyruvates + 4 ATPs + 2 NADHs
(note no O2 or CO2 involved)
Explain the overall energetics
know that the last step has a large negative G
Net energy yield is 2 ATP and 2 NADH
Be able to draw what happens to the carbon skeleton and the phosphates
Be able to figure out from the names of enzymes what type of reaction they catalyze
Sources of Glucose, Fermentation
Food Sources of glucose
Starch, polymer of glucose, bread, corn, pasta, potatoes -> glucose
Glycogen (in meat), like starch
Table sugar, sucrose, hydrolyze to glucose and fructose
High fructose corn syrup, glucose and fructose
Honey is glucose and fructose
Sweetness

fructose > sucrose, HFCS, honey, >glucose

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Trehalose, 3 glucoses, mushrooms, antifreeze -> glucose

Lactose, milk sugar, disaccharide, glucose and galactose
Mannose, in glycoproteins
Starch and glycogen, broken down by glycogen phosphorylase
Phosphorolysis, save energy in glycosidic bond
Produce glucose-1-P, then
Phosphoglucomutase converts to glucose-6-P -> glycolysis
Debranching enzyme
Fig 14-11, 14-10
Fructose controversy (Robert Lustig)
Fructose poorly taken up by muscle, brain, ~ all go to liver
In liver, glycogen content fills, then converted to fats
Correlation with metabolic syndrome
But, require high doses of fructose to see pathological effect?
humans metabolize fructose differently than rats?
If cell does aerobic metabolism - then pyruvate citric acid cycle ETC
If cell has no oxygen (muscle, RBCs have no mitos) or normally grows anaerobically
(microorganisms) then use
Fermentation, use of glucose without oxygen
Essential to recycle NADH to NAD+
Needed for step 6 in glycolysis
Animals
Pyruvate + NADH + H+ lactate + NAD+
Transfer 2e-, 2H+, regenerate NAD+
Enzyme is lactate dehydrogenase
G = -25 KJ, thus goes forward strongly
Lactate in muscle blood liver glucose
Microorganisms
Pyruvate acetaldehyde + CO2
Pyruvate decarboxylase
Acetaldehyde + NADH ethanol + NAD+ + H+
Alchohol dehydrogenase
(humans dont have pyruvate decarboxylase, but do have alcoholdehydrogenase. This allows NADH to be made from acetaldehyde, which
is why alcohol is a high calorie food)
acetaldehyde + NAD+ -> acetate + NADH
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Diet:
Sugar
Ethanol
Fat

4 calories per gram

7 calories per gram
9 calories per gram

Issues with using ethanol as biofuel (renewable?)

Start with carbohydrate? Primarily corn or sugar cane
Expensive, require water and fetilizer, fossil fuels to farm
Alternatively, use cellulose, but must convert to glucose
Still not practical
Must separate ethanol from water because fermentation is done by
Enzymes in water, distillation, at best this requires 30% of the energy in
the ethanol
Not clear if you get much more energy out of it than you put in.
Gluconeogenesis, Pentose Phosphate pathway
How to synthesize glucose and other sugars?
Animals need glucose to send energy to brain (liver -> blood -> brain)
Plants send sucrose, e.g. leaves to roots
Need to make glycosylated proteins, nucleotides, cell walls, starch
Essentially reverse glycolysis
Need to put in energy - Table 14-2, Note difference in G vs G
three steps have big -G, need to bypass these, different enzymes
Fig 14-16
1. pyruvate to PEP
2. fructose-1,6-bis-P to fructose-6-P
3. glucose-6-P to glucose
animal cells can convert protein into sugars, but cannot convert fat into sugars
protein -> amino acids, these can be converted directly into pyruvate
or into Krebs cycle intermediates and then into pyruvate
Start with pyruvate, in mitochondria because need high conc of NADH
pyruvate + HCO3- + ATP OAA + ADP + Pi
OAA + NADH malate + NAD+
malate transported to cytosol
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malate + NAD+ OAA + NADH

OAA + GTP PEP + Pi + GDP
Thus use ATP and GTP to make PEP (cost is equivalent to 2 ATPs)
but PEP pyruvate, harvest is only 1 ATP
have big -G in both directions
Note that the first reaction is carbon fixation by animals, but the CO2 is used to make
pyruvate more reactive and the CO2 is lost in the 2nd step.
If -Start with lactate, from fermentation, use different enzymes in different cell compartments
Lactate + NAD+ -> pyruvate + NADH + H+ (in cytosol)
Pyruvate enters mitochondria
Pyruvate + CO2 + ATP -> OAA + ADP
OAA + GTP -> PEP + GDP + CO2
PEP transported from mitochondria to cytosol
Difference in using lactate is in the need for NAD+ (Fig 14-19)
With pyruvate, use NADH, with lactate, make NADH
NADH is at low concentrations in cytosol
Reverse glycolysis, starting with PEP
PEP fructose-1,6-bis-P, need to use ATP and NADH
(not harvest them as in glycolysis)
fructose-1,6-bis-P + H2O fructose-6-P + Pi (FBPase-1 is enzyme)
glucose-6-P + H2O glucose + Pi

(G-6-Pase is enzyme)

Energetics - very expensive

For 2 pyruvates 1 glucose,
use 4 ATPs, 2 GTPs, 2 NADHs (equivalent to ~12 ATPs)

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Cells need 5-carbon sugars like ribose and deoxyribose, and they need NADPH
These are produced by the Pentose Phosphate Pathway (Fig 14-20)
Start with glucose-6-P and make 2 NADPHs and one ribose
Wont expect you to remember details of this pathway beyond what is shown
below (Draw the appropriate arrows)
Glucose-6-P
NADP+
NADPH
6-P-gluconate
NADP+
NADPH
CO2
ribulose-5-P
ribose-5-P
RNA etc
If you need more NADPH than ribose, then you recycle the ribose to make
more glucose-6-P
6 riboses -> 5 glucoses (6C X 5C = 5C X 6C)
Complex pathway to do this
Some of these reactions used in photosynthesis
Note that all these reactions are reversible

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