Metabolism: the entire network of chemical reactions in living cells Metabolites: small molecules which are intermediates in the degradation or biosynthesis of biomolecules.

oxidation reduction

Introduction to Metabolism

A

↓

B

↓

C

↓

D

↘

1

↓

2

↓

3

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4

α ↓ β

↓ γ

↓ δ

↓ ε

i

↑

ii

↑

iii

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iv

z

↑ y

↑ x

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↗

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Metabolic Pathways: sequences of reactions

Linear pathway - e.g. serine biosynthesis

Cyclic pathway - e.g. TCA cycle

Spiral pathway - e.g. fatty acid biosyntheis

Forms of metabolic pathways

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

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

A + B ↔ C + 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

Gibbbs Free Energy Change (G)

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 C

eq[D]

eq

Aeq

[B]eq

or 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 ×10−4 (6.31 ×10−6)

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 α-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, [α-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 [α-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.

A + B C + D E + F G

1. Sequential reactions in a pathway:

1 2 3

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 + C → D + E + F G = -31.4 kJ

A + B → D G = +30.5 kJ

C → E + F G = -61.9 kJ

2 coupled reactions-

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

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

PPi

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

ATP + H2O 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 GDP + ATP NDP kinase

NDP = Nucleotide diphosphate

- Universal electron carriers are co-enzymes: NAD+, NADP+, FMN, FAD NADH, NADPH, FMNH2, FADH2

Biological Oxidation-Reduction Reactions (REDOX)

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

- Flow of electrons is associated with free energy change:

- 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

F = Faraday constant = 96.5

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+ Ethanol + NAD+

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

E = -0.20 -0.32 = 0.12 V G = nFE = (2)(96.5)(0.123) = 23.12 kJ/mol

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 ºC

E = E + (RT/nF) ln [electron acceptor]/[electron donor] Eacteladehyde = -0.20 + (RT/nF) ln 1.0/0.1 = -0.170V ENADH = -0.32 + (RT/nF) ln 0.1/1.0 = -0.350V E = 0.180 V G = nFE = -34.74 kJ/mol

Nicotinamide ring

NAD+: Nicotinamide adenine dinucleotide NAD+ + 2e + 2H+ NADH + H+

NADP+: Nicotinamide adenine dinucleotide phosphate NADP+ + 2e + 2H+ NADPH + H+

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

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:

Sum: Glyceraldehyde 3-P + acetaldehyde → 3-phosphoglycerate + ethanol

- These co-enzymes are recycled repeatedly

- e.g. Glyceraldehyde 3-P + NAD+ → 3-phosphoglycerate + NADH + H+

Acetaldehyde + NADH + H+ → ethanol + NAD+

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)