The citric acid cycle

Stage 1 of cellular respiration

Organic fuel molecules such as glucose fatty acids and some amino acids are oxidized to yield 2 carbon fragments in form of acetyl- CoA

Stage 2 of cellular respiration

The acetyl groups are oxidized to CO2 in the citric acid cycle

Energy of the oxidation is conserved in electron carriers NADH and FADH2 by reduction

Stage 3 of cellular respiration

The reduced coenzymes are themselves oxidized giving up protons H+ and electrons

Electrons are transferred to O2 via a series of electron carrying molecules - in the respiratory chain- forming water

Pyruvate

The metabolite that links 2 central catabolic pathways- glycolysis and citric acid cycle→ a logical point of regulation that determines the rate of catabolic activity

Summarize what the citric acid cycle does

Oxidizes acetyl CoA to CO2 and H2O

Energy from the oxidation drives the synthesis of ATP

Conserving energy

Citric acid cycle is a hub if metabolism

Catabolic pathways in and anabolic out

breakdown products of many amino acids and nucleotides are intermediates in the cycle that can be fed in

How can the citric acid cycle be regulated?

Can be regulated by allosteric and covalent mechanisms to achieve homeostasis

Production of acetyl - CoA

CoA has a reactive thiol (SH) group covalent linked and critical to its role as an acyl carrier (thioester)

High standard free energies of hydrolysis→ high acyl group transfer potential

Pre-Reaction: Pyruvate→ acetyl CoA

Pyrivate is oxidized to acetyl CoA+ CO2

Occur in mitochondria and transported by MPC

By pyruvate dehydrogenase (PDH) complex- several cofactors involved: 3 enzymes and 5 coenzymes

Reaction:oxidative decarboxylation

Irreversible

NADH formed

PDH complex

3 enzymes- E1,2,3

5 coenzymes: TPP, CoA, FAD, NAD, lipoate

Reaction 1: condensation of acetyl CoA with oxaloacetate

1) OAA + Acetyl-CoA -> citrate + CoA

*) catalyzed by citrate synthase - induced fit-> example (conformational changes occur in substrate and enzyme- catalytic activity) dimer

*) "irreversible" step-> larger negative free energy change

Reaction 2: Formation of Isocitrate via cis-Aconitate

Reaction 2: Isomerization of citrate to iso-citrate

2 step process-> goes via an intermediate cis-Aconitate (not stable)-> isocitrate

Aconitase-> catalyzes hydroxylation from "correct" side of cis-aconitate

- acheived through a cofactor: iron-sulphur FeS complex (very unusual!)-> orients the correct addition of water (binding the substrate in correct orientation)

FeS→interactions points

Reaction 3: Oxidation of Isocitrate to α- Ketoglutarate and CO2

Reaction 3. Oxidated decarboxylation

Isocitrate-> alfa-keto glutarate (alfaKG)

Electrons are captured on NADH + H+

CO2 is formed

ENzyme: isocitrate dehydrogenase - "irreversible" step

The middle carboxyl group is expelled

Reaction 4: Oxidation of α-Ketoglutarate to

Succinyl-CoA and CO2

Reaction 4. aKG-> succinyl CoA

Need CoA

*) enzyme aKG DH - similar to pyruvate DH, E3 identical between PDH and aKG DH

*) Irreversible step

Reaction 5: conversion of succinyl-COA to succinate

Reaction 5: Succinyl-CoA -> Succinate + CoA

*) Succinyl CoA synthetase (tase ending-> GTP involved instead of ATP)

- The “power helices” from the α and β subunits are positioned so their dipoles (+) point toward the negatively charged phosphate, stabilizing it electrostatically.

In this case- stabilizes a phospho-histidine

*) substrate level phosphorylation of GDP to GTP (GTP is equivalent to ATP -><-)

Reaction 6: Oxidation of succinate to fumarate

Reaction 6: Oxidation of succinate->fumarate

Electrons on FADH2

*) enzyme succinate dehydrogenase

- membrane protein (mitochondrial inner membrane)

Electrons from succinate-> electron transport chain

Reaction 7: Hydration of fumarate to malate

Fumarate-> L-Malate

Water is added

*) fumarase

- stereoselective

Reaction 8: Oxidation of malate to OAA

Hydride transfer

*) malate dehydrogenase (DH)

Reversible-> unfavorable reaction

Mitochondrial matrix conditions deltaG=close to 0- reversible (low [OAA] in cells)→ pulling the reaction toward OAA

SUmmary CAC

OAA is regenerated at the end of the cycle

3 NADH, 1 FADH2, 1 GTP/ATP, 2 CO2 / PYRUVATE

Even though only 1 ATP is generated the cycle provides a large flow of electrons into the respirator chain via NADH and FADH2→ leads to the formation of 10 x more ATP

2 Pyruvate=1 GLUCOSE

Amphibolic pathway

The citric acid is an amphipholic pathway that serves in both catabolic and anabolic processes

The citric acid cycle is a hub of intermediary metabolism

Anaplerotic reactions

REactions that regenerate TCA cycle intermediates-when intermediates like oxaloacetate, malate, or α-ketoglutarate are used for making amino acids, glucose, etc., anaplerotic reactions "refill" them

Most important anaplerotic reaction

Most important: carboxylation of pyruvate by HCO3- → OAA

Catalyzed by pyruvate carboxylase

Requires ATP

Acetyl CoA stimulates the reaction to occur→ if no acetyl CoA no reaction

Requires the vitamin biotin- prosthetic group of the enzyme (activate CO2)- each subunit has a biotin molecule- totally 4

Regulation of citric acid cycle- PDH complex

PDH complex is strongly inhibited allosterically by ATP, acetyl CoA and NADH

PDH complex activity is turned off when ample fuel is available in the fatty acids and acetyl CoA, and when the c ATP is high compared to ADP and NADH higher than NAD+

Alos by P:

PDH-P -> inactive

PDH -> active

Allosterically regulated enzymes:

- Allosterically regulated enzymes:

*) PDH

*) citric synthase

*) Isocitrate DH

*) aKG DH

Metabolons

a multienzyme complexes

Dilution weakens complexes- separates enzymes

When enzymes in a pathway physically group together, they can pass substrates directly from one to the next — this is called substrate channeling→ faster reactions, more efficient, better control