The TCA Cycle

The TCA Cycle

Both glucose and fatty acids are metabolized to produce acetyl-CoA (H₃C-CO-SCoA), which is oxidized to CO₂ + H₂O

this oxidative decarboxylation takes place by means of the TCA (tricarboxylic acid) cycle, or citric acid cycle

discovered by Sir Hans Krebs in 1937 – used stoichiometric measurements from pidgeon pectoral muscles

Enzymes in the TCA Cycle

All enzymes are in mitochondria – a few exist in the cytosol where they have other functions

(1) Citrate Synthase – mitochondria only – regulation: citrate (-), ATP (-)

(2) Aconitase – cytosol and mitochondria (different isozymes); Fe/S center – favors reverse reaction (mass action drives fwd)

(3) Isocitrate Dehydrogenase – cytosol and mitochondria (different isozymes) – regulation: NADH (-), ADP (+), Ca²⁺(+)

unidirectional – committed to forward catalysis, rate controlling step

(4) a -Ketoglutarate Dehydrogenase – mitochondria only – regulation: NADH (-), succinyl CoA (-), Acetyl CoA (-), Ca²⁺(+)

(5) Succinyl CoA Synthase – mitochondria only – produces GTP (made into ATP by nucleotide diphosphokinase)

(6) Succinate Dehydrogenase – mitochondria only (bound to inner membrane) – uses FAD as coenzyme; FADH₂(-)

(7) Fumerase – cytosol and mitochondria (cannot cross inner membrane)

(8) Malate Dehydrogenase –cytosol and mitochrondria – favors reverse reaction – used during gluconeogenesis; NADH(-)

Primary regulation occurs at citrate synthase, isocitrate dehydrogenase, and a -ketoglutarate dehydrogenase

the most crucial regulator is NAD⁺/NADH ratio - rate of cycle depends primarily on rate of NADH removal (e^- transport)
ratio is 700:1 in liver cytosol, 10:1 in mitochondria (compare NADP⁺/NADPH ratio of 0.01:1 – so, in contrast to NAD⁺ which oxidizes by accepting an H^-, NADPH usually reduces other molecules by donating its H^-)
each NADH can be oxidized to produce 3 ATP; each FADH₂ produces 2 ATP – 3 NADH + 1 FADH₂ + 1 GTP = 12 ATP

Anapleurotic ("Refilling") Reactions

In the muscle, the TCA cycle is closed (otherwise it would not have been discovered by stoichiometry). But, in the liver, several intermediates are used for biosynthetic reactions.

Anapleurotic reactions "refill" the cycle – there are four of them:

(1) pyruvate carboxylase – most important reaction – pyruvate + CO₂ + ATP Þ oxaloacetate + ADP

requires biotin, like most carboxylation enzymes
regulated by acetyl CoA (+), which brings subunits together – so activated during fatty acid oxidation

(2) glutamate dehydrogenase – glutamate + NAD⁺ Þ a -ketoglutarate + NADH + H⁺ + NH₃

reaction is called oxidative deamination – free NH₃ becomes urea

(3) transaminases – all use vitamin B₆ (pyridoxal phosphate) and use coreaction a -ketoglutarate Þ glutamate

aspartate aminotransferase (AST) – aspartate Þ oxaloacetate – mitochondria and cytosol

alanine aminotransferase (ALT) – alanine Þ pyruvate – mitochondria only

(4) propionyl CoA carboxylase/mutase – makes succinyl CoA from propionic acid (H₃C-CH₂-COOH) Þ propionyl CoA

propionic acid comes from diet (swiss cheese), branched-chain amino acids, or odd-number fatty acids

propionyl CoA carboxylase adds CO₂ to second carbon to make methylmalonyl CoA (requires biotin)

propionyl CoA + ATP + CO₂ Þ methylmalonyl CoA + ADP

methylmalonyl CoA mutase moves COO^- group to terminal carbon, making succinyl CoA (requires B₁₂)

this is one of the only two known uses of vitamin B₁₂ (the other is methionine metabolism)
methylmalonyl CoA appears in urine if B₁₂ is deficient (methylmalonic Acidurea)

isoleucine, valine, methionine Þ proprionyl CoA

Pyruvate Dehydrogenase Complex (PDC)

Pyruvate (the end result of glycolysis) is made into acetyl CoA by the irreversable pyruvate dehydrogenase complex

occurs in the mitochondria only – net reaction: pyruvate + NAD⁺ + CoA Þ AcCoA + NADH + H⁺ + CO₂

a -ketoglutarate dehydrogenase complex (in TCA cycle) is very similar

It involves more than 150 separate proteins – five important ones:

(1) E₁(dehydrogenase) – requires thiamine (vitamin B₁), inactivated by phosphorylation (by E₁ kinase)

(2) E₂ (transacetylase) – contains lipoic acid adduct

(3) E₃ (dihydrolipoyl dehydrogenase) – oxidizes lipoic acid on E₂ and produces NADH in the process

(4) E₁ kinase – inhibits E₁ – regulated by ATP (+), NADH (+), Acetyl CoA (+); also pyruvate (-), ADP (-), Ca⁺⁺ (-)

thus active when energy levels are high to turn off the pyruvate carboxylation

(5) E₁ phosphatase – activates E₁ – regulated by Ca⁺⁺ (+), insulin (+) – but not cAMP dependent

The reaction takes place in four steps:

(1) Two carbons bind the thiazolium ring of vitamin B₁ of E₁

(2) Carbons are transferred to lipoic acid sulfur on E₂

(3) Acetyl CoA is released from E₂

(4) E₃ oxidizes E₂ and produces NADH

TCA Cycle Regulation – an example

During fasting: [glucose] ß , [insulin] ß , [glucagon] Ý

low insulin causes digestion of muscle protein into 20-amino acid units – alanineÞ pyruvate, glutamateÞ a -ketoglutarate

adipose tissue triglycerides are converted into free fatty acids and glycerol; b -oxidation produces NADH and acetyl CoA

high acetyl CoA levels (from fatty acid oxid.) inactivate PDC, causing pyruvate (from muscle alanine) not to enter TCA

rather, pyruvate Þ oxaloacetate (via pyruvate carboxylase), then to malate (backwards along TCA)

malate is shuttled out of the mitochondria, then Þ oxaloacetate Þ glucose

high NADH levels (from fatty acid oxid.)inhibit the TCA cycle by inhibiting isocitrate and a -ketoglutarate dehydrogenases

the TCA cycle sacrifices precious carbon by release of CO₂, preventing gluconeogenesis
since acetyl CoA cannot enter TCA, they are made into ketone bodies instead (activated by low insulin)

low ADP levels (from fatty acid oxidation) inihibit isocitrate dehydrogenase, causing buildup of isocitrate

this causes buildup of citrate, which inhibits citrate synthase