1

BIOC 301 - Lecture 2 Olson - Fundamentals of Metabolism and Energy Coupling

(LPOB-Part II, pp. 501-504, LPOB-13, pp. 505-528, 532-535)

A. Metabolic and functional hierarchy of cells:

Six key functions:
3. Gene expression

(genes to codons)

DNA
(gene storage)

mRNA

synthetic products
(sugars, amino acids,
fatty acids, etc.)

4. Proteins
(structural and
enzymes)

Translation
(codons to proteins)

6. Anabolism

Rb

precursors
and intermediates

2. Replication
(semi-conservative)
"Chemical energy of cells"

5. Catabolism
Fuels:
sugars
fats
amino acids
O2

Acids
or
CO2, H2O
light

Cell division

choroplasts
(plants and blue
green algae)

outside
1. Cell membrane (separation from environment and storage of energy by ion gradients )

Transcription

inside

1) Compartmentalization by a
lipid bilayer and selective
permeation of metabolites
and products.
2) Gene storage and
replication.
3) Gene expression (DNA
mRNA protein:
transcription and translation).
4) Gene products: structural
proteins and enzymes that
catalyze all the other
processes.
5) Catabolic energy
production through ATP,
NADH, and H+ gradients.
6) Anabolic use of ATP,
NADPH and H+ gradients.
Perhaps the most crucial
phenomena involve the
precise regulation of all six
of these processes.

B. The basic catabolic pathway involves the oxidation of glucose:

C 6H12 O6 + 6O2

6CO 2 + 6H2 O

Go ' =-2840 kJ/mol

(Conserved in the form of ATP)

1. Catalysis: The "burning" of sugars does not

ordinarily occur by simply heating. Glycolysis and the
TCA cycle carry out this process in seconds,
conserving ~40% of the liberated free energy for
metabolic use in the form of 38 ATPs (see LPOB-14, 16;
Table 13.4; Fig. 16-1).

2. Coupling: The ATP formed from the complete oxidation of glucose can diffuse away from the
catabolic enzymes and organelles toward synthetic enzymes and organelles (particularly the nucleus and
the endoplasmic reticulum).
Glutamine synthetase again: amide bond formation driven by ATP hydrolysis (Lecture 1, pp 1-3; LPOB, Fig. 13-18).

2
glutamate
CO2
H3N C H

ammonia
+

CH2
CH2

H2N

adenosine triphosphate (ATP)

O

NH3

N
O P O P O CH2 O
OH O
O

N
(an adenine
nucleotide)

OH OH

glutamine synthetase

CO2
H3N C H

CH2
CH2
C
NH2
O
glutamine

O
O

H2N

inorganic phosphate

O P O P O CH2 O N
O
O

P O
OH

OH OH
adenosine diphosphate( ADP)

3. Regulation: If plenty of ATP is available, oxidation of glucose is shut off and its storage or conversion
into other metabolites is turned on and vice versa. In living cells, glucose is not oxidized unless energy
for growth, movement, or other activities is needed. In the case of glutamine synthetase, high ATP turns
on its activity because this reaction is a key first step in the synthesis of the building blocks for nucleic
acids and proteins.
high ATP
activates
storage
and inhibits
breakdown

high ADP
inactivates
storage and
activates
breakdown

high ADP
activates

high ATP
inactivates

"oxidation"

6CO2 + 6H2O

C6H12O6 + 6O2
ADPi, Pi

ATP
high ATP
activates

Storage polysaccharides,
Glycogen in animals
(or eventually fat)

high ADP
inactivates

NH3 + glutamate
glutamine
"biosynthesis"
nucleic acids
amino acids

and many other

anabolic pathways

NH3 in urine

C. Chemical potentials, equilibrium constants, and free energy changes (LPOB-13, pp. 507-511)
1. The A+B hypothetical reaction:

A + B

The free energy change for this reaction is:

G = products reactants

C +D

3
2. The chemical potential, , for reactant A is: A = A + RTln(aA) where:
a. A is the standard chemical at 1 molar concentration of A. It is the intrinsic potential of the
compound A to react with the other reactant molecule, B. Each molecule represents a microscopic
system of charges and bonds and exhibits a certain characteristic potential to react (hydrolyze in the
case of ATP) and change its bonding pattern. The A term is a measure of the instability of this
system with respect to that of other the reactant and product molecules.
b. RTln(aA) or RTln[A] is an extrinsic term that reflects the decrease in free energy due to dilution.
The higher the concentration of the reactant, the greater is its potential to react (i.e., it is more
concentrated and has less entropy). This term is analogous to the number of electrons on a capacitor
- the more charges, the greater the voltage and the greater the potential for electrical work. By
analogy, at higher concentrations, high-energy phosphate compounds will have an even greater
potential for hydrolyis. (Analogous to P=(n/V)RT more pressure and potential for work at higher
concentration (n/V).)
3. General free energy change for the reaction
G = products reactants = C+RTln[C] + D+RTln[D] - (A+RTln[A] + B+RTln[B])
[C][D]
G = G o + RT ln
[A][B]

where G = difference in standard chemical potentials, (C+D) - (A+B).

Note that when G < 0, it is the amount of free energy in kJ/mol released at temperature T when 1 mole
of reactants is converted to 1 mole of products at the given [A], [B], [C], and [D], which are kept at the same
level during the conversion. When G > 0, free energy must be added to force the reaction to occur. G is
a hypothetical value for in vitro reactions, but it is also applicable in living cells and organisms where, in the
steady state, the ratio of concentrations is often relatively constant.
Note: the gas constant: R = 8.314 J/mol-K or 1.987 cal/mol-K (memorize both)
4. Two key conditions that give meaning to the general free energy equation:
a. At equilibrium, no free energy is available, and by definition the reaction is complete.
" [C]'[D]' %
' where [A]', [B]', [C]', and [D]' represent the equilibrium
G = 0 = G + RT ln$
# [A]' [B]' &
concentrations of reactants and products. Note that:
# [C]"[D]" &
# [C]'[D]' &
o
Keq = %
( or RTLnK eq
( and thus: G = RT ln %
$ [A]'[B]' '
$ [A]"[B]" '

# G o &
(
(
$ RT '

The key relationships are: G o = RT ln K eq or K eq = exp %%

Thus, the measured equilibrium constant defines the standard free energy change, G, at a given
temperature and vice versa.

4
b

At 1 M standard conditions: G = G and is equal to the free energy change of taking 1 mol of
reactants to 1 mol of products when [A] = [B] = [C] = [D] = 1 M. It is defined experimentally as

G = RT ln K eq

4. Under steady-state, cellular conditions G is given by the general equation (LPOB-13, pp. 518-523):
" [C][D]%
' ; and, as a result, the free energy available can be much different than
G = G + RT ln $
# [A][B]&
G. As discussed below, the free energy released by the hydrolysis of ATP is used to couple reactions
in biological systems and can vary greatly with cellular conditions.
ATP + (H2O)

G(25C , pH 7.0) = - 30.5 kJ mol-1

ADP + Pi

" [ADP][P ]%
i '
where: G = G + RT ln $
# [ATP] &

Remember: G must be < 0 for a reaction to occur.

In cells with plenty of glucose and a high "energy

potential," the concentrations of reactants and products are:
[Pi] = 1x10-2 M
[ATP] = 1x10-2 M (high)
[ADP] = 1x10-5 M (low)

In cells that are starved of glucose and at a low "energy

potential," the concentrations of reactants and products are:
[Pi] = 1x10-2 M
[ATP] = 1x10-5 M (low)
[ADP] = 1x10-2 M (high)

Under these conditions G = - 59 kJ mol-1 at 25C

(Lots of "driving force" for hydrolysis and synthesis)

Under these conditions G = -25 kJ mol-1 at 25C

(Less "driving force," because RTln term is 0)

D. Special nature of ATP and other "high energy" phosphate compounds (LPOB-Fig. 13-11):
Two kinetically stable,"high
energy" phosphoanhydride bonds.

O
High charge
density prevents
hydrolysis by
H2O.

-O

-O

NH2

-O

Mg2+

Divalent cation
neutralizes negative charges.

Key properties of ATP:

-O

N
CH2
H
H

OH

N
N

H
H
OH

Binds to highly conserved

nucleotide binding sites
on proteins and enzymes.

ATP(Mg2+)

(1) "High energy" character is due to electrostatic repulsions of negative charges, more resonance stabilization
in the products, and greater solvation of products. Note that all anhydrides hydrolyze readily with G << 0
(very negative and favorable).
(2) Mg2+ is usually bound to ATP and ADP.
(3) The phospho-anhydride bonds are kinetically stable and usually require a coupled chemical reaction for
hydrolysis. Note that arsenate anhydride bonds are unstable in water and hydrolyze spontaneously. However,
inorganic arsenate, HAsO4=, "looks" just like HPO4= and is incorporated into ADP as ADP~Asi, which then
breakdowns before any enzymatic coupling can occur. This spontaneous hydrolysis is why arsenate is a poison.

5
Table 1: Phosphate ester and anhydride hydrolysis free energies at pH 7, 25C (LPOB-Table 13-6):
Nucleotide
phosphates

G'
(kJ/mol)

High energy metabolites

phosphoenol pyruvatea

low energy
metabolites

G'
(kJ/mol)

G
(kJ/mol)

-61.9

ATPADP,Pi

-30.5

glucose-1-P

-20.9

1,3-diphosphoglycerate

-49.3

ATPAMP, PPic

-45.6

fructose-6-P

-15.9

acetyl phosphate

-43.1

PPiPi, Pic

-19.2

glucose-6-P

-13.8

-43.0

glycerol-1-P

-9.2

phosphocreatine
a

Note that phosphoenol pyruvate and 1,3-diphosphoglycerate are intermediates in glycolysis that phosphorylate ADP to
ATP.

Phosphocreatine is the "high energy" phosphate buffer or reservoir in muscle tissue to maintain ATP levels during stress.

ATP hydrolysis to AMP and the subsequent hydrolysis of pyrophosphate to two inorganic phosphates gives a net G' =
-64.8 kJ/mol at room temperature pH 7. This "trick" is used to drive unfavorable reactions (i.e. acylation of free fatty
acids to acylCoA thiol esters (LPOB-13, p. 505-506)).

Examples of coupling to form or utilize ATP:

1. Pyruvate kinase reaction from the last steps in glycolysis (LPOB-13, pp. 522-523; LPOB-14, p. 554-555):
(a) ATP formation from Pi and ADP is unfavorable:

ADP + Pi

G'= + 30.5 kJ/mol

ATP + (H2 O)

(b) Hydrolysis of phosphoenol pyruvate is very favorable (G'=-61.9 kJ/mol) due to enolate
isomerization:
O
O

C
(-)O

C
(-)O

H+

O-

C
-O

O(-)

G' = -46 kJ/mol

The driving "force" for hydrolysis
of PEP is enolate isomerization
to the more stable ketone.

H
inorganic phosphate, Pi

O
O

C
-O

C
C

-O

carbanion to keto group

(strong nucleophile)

(-)

O-O

enolate intermediate

phosphoenol pyruvate
(PEP)

HO

C
H

G' = -16 kJ/mol

H+

Pyruvate

C
H

H
H

(c) Thus, the net pyruvate kinase reaction is reaction is favorable:

PEP + ADP

ATP + pyruvate

G'= - 31.4 kJ/mol

6
2.

Glutamine synthetase reaction: amide bond formation driven by ATP hydrolysis (see p.2, LPOB, Fig. 13-18).
(a) Amide formation is unfavorable.

glutamate + NH3

G'= + 14.2 kJ/mol

glutamine + (H2O)

(b) ATP hydrolysis is very favorable - the driving force.

ATP + (H2O)

G'= - 30.5kJ/mol

ADP + Pi

(c) The sum, (1) + (2), is a favorable reaction. The enzyme is able to use efficiently the chemical energy
"stored" in ATP to form a peptide bond.

glutamate + NH3 + ATP

G'= - 16.3 kJ/mol

glutamine + ADP + Pi

E. Oxidation of sugars and other carbon compounds - the role of NADH as a hydride carrier.
1. Biological oxidations of alkanes, alcohols (hydrated alkenes), and ketones (LPOB Figs. 13-9, 13-10, p. 520
and LPOB-13, pp. 532-535, Fig. 13-24*).
2e-,2H+

H2O
H C H

2e-,2H+

H C OH

C
2e-,2H+
Usually
FAD or FMN
(succinateDH
or FA oxidation)

H2O

(hemi-acetal)

simple
hydride, Htransfer to
NAD+

H2O 2e-,2H+

or CO2

hydride, H- transfer to
NAD+ and more complex
mechanisms for ketones
(normally -keto acids
use thiamin PPi)

Electrons are
eventually transported
to dioxygen, O2
H
H

C
H

O C

C
NH2

H+ +

pro-S

pro-R

NH2

R
NADH (absorbance peaks - 260 and 340 nm

R
(absorbance peak - 260 nm
NAD+ no fluorescence)

fluorescence at ~450 nm)

2. The hydride (H-) transfer reaction with NAD (nicotinamide or niacin):

pro-S

pro-R

H
O C

NH2

H
+

NH2
H+ +

N
R
(absorbance peak - 260 nm

NAD+ no fluorescence)

R
NADH (absorbance peaks - 260 and 340 nm
fluorescence at ~450 nm)

7
+

3. Structure and properties of NAD (usually involved in catabolism) and NADPH (LPOB Fig. 13-24):

4. Stereochemistry of hydride transfer - note that there is no equilibration with solvent protons or H atoms.
(See also Cahn-Ingold-Prelog system, LPOB-1, pp. 16-19 (rectus,"right" versus sinister, "left"; -OCH3 > -OH > -NH2 > CO2H > -CHO > -CH2OH > -CH3 > -H); your organic textbook for a discussion of prochiral molecules; and LPOB, Box 163, pp. 648).

a. Experimental observations:
(1.04D/mol in acetaldehyde)

(0.96D/mol in ethanol)

OH

ADH
D

+ NADH + H+

C
D

CH3

CH3
O
O
-OH

H + NAD+

CH3

Distill and purify

OH
pro-S

O
Cl S
O

D
CH3

SN2

NAD+
ADH

D H
O
O S
O

HO-

NADH, H+

CH3

CH3
D

O
O

ADH

C
+ NADD + H+

CH3

H
NAD+

CH3

Form tosylate and

invert stereochemistry

CH3

OH

pro-R

CH3

(0.77D/mol)

(0.00D/mol)

Transfer is stereospecific with respect to the two H atoms of ethanol.

CH3

(0.77D/mol in acetaldehyde)

Hydride transfer is
direct (no loss of label)

8
b. Addition and subtraction of the hydride from different sides of the nicotinamide ring (LPOB-Table 13-8):
T

ADH
CH3CT2OH + NAD+
N
R

O
CH3CH

N
R

Control
ADH

C O
H3C

CH3CCO2pyruvate

+ H C T
CH3

C
NH2

CH2OPi

+ HO C H

N
R

NH2

lactateDH
alcoholDH

N
NH2

CO2-

NADH

+ HO C T

N
R

CH2OH

OH

pro-R, re-side
A side addition

O H

GAPDH
glycerolPDH

lactateDH (LDH)

-glycerol-PDH
NH2

pro-S, si-side
B side addition

isolate NADH(T)
dihydroxyacetone-Pi

O
C

NH2

CH3
L-lactate

5. Properties of L-lactate dehydrogenase (LDH) (LPOB Fig.13-10, p. 533-535; Fig. 13-24; LPOB-14, p. 563-565; LPOB-15, p.
602)

a. Reaction:

H+

CO2
NADH

+ O

CO2 NAD+

+ HO C

CH3
pyruvate

CH3
L-lactate

b. Inhibition by excess substrate in the heart enzyme (reaction of pyruvate with NAD+, both are oxidized
substrates):
His195

CH2

CH2

acidity of carbon atom

to the keto group

HN

(-)

H2N

(+)

O-

C NH2

(+)

N R

NH2

HN

H2C

CH2

(+)
N
H

HN

Inhibited pyruvate-NAD+
complex

O C
(-) O
H2N
(+)
C NH2

His195

CH2
HN

H2C

(+)

H2C

HN

C NH2

Arg171

HN

Arg171

(-) O

H2N

NH2

proR Hydride, re-face

Arg171

His195

proR Hydride, re-face

HO
H

(+)
N
H

HN

H
C

enediolate
intermediate

His195

C O

proR Hydride, re-face

C NH2

N
H

carbanion

(+)
H

H
C

H
(-)
O

R
O C
(-) O
H2N
(+)
C NH2
Arg171

HN

H2C

proR Hydride, re-face

C NH2

(+)

9
c. Active site (with Rossman folds):

d. Probable orientation of substrates and the mechanism for hydride transfer (note the product is L-lactate):
Reaction mechanism for LDH

His195

CH2
HN

Reduction
+N

oxidation
N

CH3

H
CH3

(-)
H2N

(+)

C
HN

H O

H2C

CH2
HN

Arg171

His195

proR Hydride, re-face

H+

C NH2

H2N

NH2

(-)
(+)

C
Arg171

HN

H2C

proR Hydride, re-face

H
O

N R
H

C
O

H
O

NH2

C NH2