Introduction to Metabolism

Metabolism: the entire network of chemical reactions in living cells

Metabolites: small molecules which are intermediates in the degradation

or biosynthesis of biomolecules.

reduction

oxidation

A
z

i
1

ii
B
2

iii
C

D
iv
4

Metabolic Pathways: sequences of reactions

Forms of metabolic pathways
Linear pathway
- e.g. serine biosynthesis

Cyclic pathway
- e.g. TCA cycle

Spiral pathway
- e.g. fatty acid
biosyntheis

Single step vs multistep pathways

Energy carriers:
- ATP
- NADH/NADPH

Metabolic pathways are regulated

Feedback inhibition

Feed-forward activation

Overview of anabolic pathways

- Autotrophs vs heterotrophs
- Photoautotrophs vs chemoautotrophs

Overview of catabolic pathways

Cellular compartmentation of metabolic pathways

gluconeogenesis

Gibbbs Free Energy Change (G)

G = a measure of available energy from a reaction
G = free energy change under standard conditions
[standard conditions: 1 atm; 25C; pH = 7;
1 M concentration for all reactants and products]
G is an indication of the spontaneity of a reaction

A+BC+D
- Sign (+/-) indicates direction of a reaction
- (+) endergonic reaction: requires an input of free energy; energetically
unfavorable
- (-) exergonic reaction: releases free energy (spontaneous); energetically
favorable
- At equilibrium, G = 0
Magnitude of G is an indication of amount of work that can be done by
chemical reaction before it reaches equilibrium

How to determine the actual free energy for a reaction?

Consider the reaction A + B C + D

G = G + RT ln

C [D]
A [B]

R = gas constant (8.315 J/mol K)

T = temperature in K (25 C = 298 K)
At room temperature, RT = 2.478 kJ/mol
At equilibrium, there is no force driving the reaction in either direction,
thus G = 0. Then the above equation becomes:
0 = G + RT ln

or

C eq[D]eq
A eq[B]eq

G = RT ln K eq (K eq = Equilibrium constant)

Exercise
1. Consider the following reaction:
Fructose-1,6-bisphosphate DHAP + 3-PG

G = +24.0 kJ/mol
[3-PG] = 6.31 10-6 M
[DHAP] = 1.58 10-4 M
What is the lowest concentration of Fructose-1,6-bisphosphate which will
allow this reaction to proceed forward at room temperature?
Let [Fructose-1,6-bisphosphate] be X M:
G

= G + RT ln

C [D]
A [B]

= 24 + 2.478 ln

1.58 104 (6.31 106)

X

At equilibrium, G = 0, solving for x:

x = 1.60 10-5 M
In order for the reaction to proceed forward, [Fructose-1,6-bisphosphate] must be greater than
1.60 10-5 M so that G < 0.

2. The following reaction is catalyzed by the enzyme L-glutamate-pyruvate

aminotransferase:
L-glutamate

pyruvate

-ketoglutarate

L-alanine

At 25 C, the equilibrium constant for the reaction is 1.11. Predict which

direction will the reaction proceed if the concentrations of the reactants and
products are
[L-glutamate] = 30 M
[pyruvate] = 0.33 mM,
[-ketoglutarate] = 16 mM
[L-alanine] = 6.25 mM

(b) Which of the following conditions would cause this reaction to go forward?
i. Adding more of the enzyme
ii. Increasing both [L-glutamate] and [pyruvate] to 20 mM
iii. Decreasing both [-ketoglutarate] and [L-alanine] to 20.0 M

How could unfavorable reactions proceed in a cell?

Every metabolic pathway must be an energetically-favored process.
1. Sequential reactions in a pathway:
1

A+B C+DE+FG
G1= G1= -13 kJ/mol; G2= +20 kJ/mol; G1= -10 kJ/mol
G (A +B G) = -3 kJ/mol

2. Coupled reactions in a single step:

A+B+CD+E+F

G = -31.4 kJ

2 coupled reactions-

A+BD
CE+F

G = +30.5 kJ
G = -61.9 kJ

ATP - the universal energy currency of the cell

Hydrolysis of ATP
-

ATP + H2O ADP + Pi

ATP + H2O AMP + PPi
Cleavage of phosphoanhydride bonds

Hydrolysis of phosphoanhydride bonds in ATP is energetically favorable

(- G is large and negative)

Three factors contributing to the large amount of energy released

during ATP hydrolysis
-

ATP + H2O ADP + Pi

ATP + H2O AMP + PPi
PPi + H2O 2Pi

PPi

3. Solvation effects
- ADP and inorganic phosphate (Pi) or AMP and pyrophosphate (PPi) are better
solvated than ATP
- Solvated ions are electronically shielded from one another

Actual free energy change for ATP hydrolysis

G = - 30.5 kJ/mol under standard conditions (1M for all

reactants and products)

G of ATP hydrolysis in living cells is very different

[ATP], [ADP], and [Pi] are not identical and much lower than
the standard conditions

In human erythrocytes, [ATP] = 2.25 mM, [ADP] = 0.25 mM ,

and [Pi] = 1.65 mM

G is much more negative than G (-52 kJ/mol in

erythrocytes) and the driving force is much larger

ATP is stable in living cells

ATP is thermodynamically unstable

ATP is kinetically stable

The activation energy is huge (200-400 kJ/mol) for uncatalyzed

hydrolysis of ATP

No spontaneous hydrolysis and donation of phosphoryl group

Specific enzymes are required and regulated for the disposition of

energy carried by ATP

Phosphoryl group transfer by ATP

- Hydrolysis of ATP could drive endergonic biosynthetic reactions
Example:
-

Glutamate + NH4+
ATP + H2O

Glutamine + H2O (G = +14 kJ/mol)

ADP + Pi + H+
(G = -32 kJ/mol)

A two-step process
ATP is covalently involved in the phosphoryl group transfer
Phosphoryl group is first transferred to the substrate for activation
The phosphate-containing moiety is displaced

Production of ATP by phosphoryl group transfer

Metabolites with high phosphoryl group transfer potential can donate a
phosphoryl group to ADP to form ATP

Example:

Nucleotidyl group transfer

Coenzyme A

CoA-SH

Thioesters

- Another class of high energy compounds

- A sulfur atom replaces the oxygen atom in the ester bond

- With large free energy of hydrolysis

Production of GTP (ATP) through hydrolysis

of thioesters:

GTP + ADP

NDP kinase

GDP + ATP

NDP = Nucleotide diphosphate

Biological Oxidation-Reduction Reactions (REDOX)

- Oxidation: loss of electrons; Reduction: gain of electrons

- Universal electron carriers are co-enzymes:
NAD+, NADP+, FMN, FAD
NADH, NADPH, FMNH2, FADH2
- Flow of electrons is associated with free energy change:

F = Faraday constant = 96.5

- Electron flow is favorable from molecules of lower reduction potential

to molecules of higher reduction potential
- Energy released from electron flow can be used to make ATP

Reference half-reaction:

2H+ + 2e- H2 (E = 0 V)
[1 M H+ and 1 atm H2
pH 0]

Electrochemical cell measurement of electromotive force (emf)

Exercise
1. Calculate the G for the following reaction using the information in the table above
Acetaldehyde + NADH + H+

Solution:
The relevant half-reactions and their E are:
Acetaldehyde + 2H+ + 2e- ethanol

E = -0.20 V

NAD+ + 2H+ 2e- NADH + H+

E = -0.32 V

= -0.20 -0.32

=
=
=

= 0.12 V

nFE
(2)(96.5)(0.123)
23.12 kJ/mol

Ethanol + NAD+

Exercise
2. Calculate the G value for the above reaction if acetaldehyde and NADH are
present at 1.0 M while ethanol and NAD+ are present at 0.1 M at 25 C

Eacteladehyde

-0.20 + (RT/nF) ln 1.0/0.1 =

-0.170V

ENADH

-0.32 + (RT/nF) ln 0.1/1.0 =

-0.350V

0.180 V

nFE

-34.74 kJ/mol

(RT/nF) ln [electron acceptor]/[electron donor]

NAD+ and NADP+: water soluble electron carriers (coenzymes-cosubstrates)

NAD+: Nicotinamide adenine dinucleotide
NAD+ + 2e + 2H+ NADH + H+
NADP+: Nicotinamide adenine dinucleotide phosphate
NADP+ + 2e + 2H+ NADPH + H+
Nicotinamide
ring

In many cells and tissues:

- NAD+ (oxidized): NADH (reduced) ratio is high
- NADPH (reduced): NADP+ (oxidized) ratio is high
- NAD+ functions in oxidation (catabolism)
AH2 + NAD+ A + NADH + H+
- NADPH functions in reduction (anabolism)
A + NADPH + H+ AH2 + NADP+
No net production or consumption of NAD or NADP in redox reactions:
- e.g.
Sum:

Glyceraldehyde 3-P + NAD+ 3-phosphoglycerate + NADH + H+

Acetaldehyde + NADH + H+ ethanol + NAD+
Glyceraldehyde 3-P + acetaldehyde 3-phosphoglycerate + ethanol

- These co-enzymes are recycled repeatedly

Flavoproteins:
- FMN: Flavin mononucleotide; FAD: Flavin adenine dinucleotide
- Flavin nucleotide is derived from vitamin riboflavin (B2)
- Accept 1 or 2 electrons (H atoms)
- Fully reduced forms: FMNH2 and FADH2 (Amax = 360 nm)
- Partially reduced forms: FMNH and FADH (Amax = 450 nm)

- Involved in greater diversity of redox reactions

- Bound to enzymes as prosthetic groups (Coenzymes)