Lecture 30- The Citric acid cycle II

The citric acid cycle

-takes place over 8 steps and requires several unique enzymes

-these reactions occur after pyruvate has been converted to acetyl-CoA and involves various isomerization’s, redox reactions, decarboxylation’s and hydrations

step 1 of the citric acid cycle

-a condensation reaction where a new C-C bond is formed between oxaloacetate and acetyl-CoA to generate citrate

-this reaction is catalyzed by citrate synthase and results in a 6C molecule being formed from a 2C and a 4C molecule

Step 2 of the citric acid cycle

-citrate is isomerized to isocitrate by an enzyme known as aconitase

-aconitase requires Fe²⁺ as a cofactor and performs a reaction where a symmetrical (and achiral) citrate molecule is converted into an asymmetrical, chiral compound

-this reaction involves the movement of an -OH group from carbon C2 to carbon C1

step 3 of the citric acid cycle

-oxidative decarboxylation of isocitrate (a 6C compound) to ɑ-ketoglutarate (a 5C compound) by isocitrate dehydrogenase

-this two step reaction first involves the oxidation of isocitrate to oxalosuccinate; this reaction generates a molecule of NADH

-in the second step, oxalosuccinate is decarboxylated to ɑ-ketoglutarate; the ɑ-ketoglutarate is a versatile metabolite that can be used in several metabolic pathways

step 4 of the citric acid cycle

-oxidative decarboxylation reaction of ɑ-ketoglutarate in the presence of Co-A

-this reaction is catalyzed by the ɑ-ketoglutarate dehydrogenase complex, and is similar to the conversion of pyruvate to acetyl-CoA

-the ɑ-ketoglutarate hydrogenase complex uses TPP and lipoic acid as cofactors to decarboxylate ɑ-ketoglutarate and generate succinyl-CoA (a 4C compound)

-at this point, 2 molecules of CO₂ have been generated and 2 molecules of NADH have been generated

-decarboxylation in vivo effectively makes these reactions irreversible

step 5 of the citric acid cycle

-the thioester bond of succinyl-CoA is hydrolyzed to produce succinate and CoA-SH; an accompanying reaction is the phosphorylation of GDP to GTP

-this reaction is catalyzed by succinyl-CoA-synthetase

-a synthetase is an enzyme that creates a new covalent bond and requires the direct input of energy from a high energy phosphate molecule (typically from ATP)

-the energy required to phosphorylate GDP to GTP comes from the hydrolysis of the succinyl-CoA thioester; GTP is then used by nucleotidediphosphate kinase (NDPK) to generate ATP from ADP (an example of substrate-level phosphorylation)

Step 6 of the citric acid cycle

-succinate is oxidized to fumarate; this reaction is catalyzed by succinate dehydrogenase

-this reaction occurs at the inner mitochondrial membrane (IMM) and uses FAD as an oxidizing agent; as a result, FAD is reduced to FADH₂ during this reaction

-like NADH, FADH₂ is a strong reducing agent that can be used in subsequent reactions, including oxidative phosphorylation to generate ATP

-succinate dehydrogenase will be encountered again when we cover oxidative phosphorylation

step 7 of the citric acid cycle

-water is added across the double bond of fumarate by fumarase to generate L-malate

-malate is a chiral molecule that has 2 enantiomers, but only 1 is produced here (L form)

step 8 of the citric acid cycle

-final step, involves the regeneration of oxaloacetate from L-malate; this is catalyzed by malate dehydrogenase

-in this reaction, L-malate is oxidized to oxaloacetate, while another NAD⁺ molecule is reduced to NADH

net rxn for the citric acid cycle

Acetyl-CoA + 3 NAD⁺ + FAD + GDP + P_i + 2 H₂O → 2 CO₂ + CoA-SH + 3 NADH + 3 H⁺ + FADH₂ +GTP

the total eventual production of ATP from pyruvate

4 NADH → 10 ATP (2.5 ATP for each NADH)

1 FADH₂ → 1.5 ATP (1.5 ATP for each FADH₂)

1 GTP → 1 ATP (1 ATP for each GTP)

total = 12.5 ATP per pyruvate or 25 molecules of ATP per glucose

the reaction of pyruvate to acetyl-CoA

-is exergonic (ΔG^o’ = -33.4 kJ/mol)

-the citric acid cycle itself is exergonic (ΔG^o’ = -44.3 kJ/mol)

1 strongly endergonic rxn in the citric acid cycle

-the oxidation of L-malate to oxaloacetate (ΔG^o’ = +29.2 kJ/mol)

-this endergonic rxn is coupled to a strongly exergonic reaction: the condensation of acetyl-CoA and oxaloacetate to produce citrate (ΔG^o’ = -32.2 kJ/mol)

in order for the cell to function properly

-the citric acid cycle must be regulated

-there are 4 primary control points associated with the citric acid cycle in which the enzymes of these reactions are regulated by various metabolites

pre-cycle regulation

-the pyruvate dehydrogenase complex is regulated by the binding of ADP, ATP, acetyl-CoA or NADH

-here, ADP (which signals that energy stores in the cell are low) will activate the pyruvate dehydrogenase complex, while the other 3 metabolites will inhibit the enzyme

3 regulated enzymes in the citric acid cycle

-citrate synthase

-isocitrate dehydrogenase

-ɑ-ketoglutarate dehydrogenase complex

citrate synthase

-is an allosteric enzyme that is inhibited by ATP, NADH, succinyl-CoA and citrate (its own product)

isocitrate dehydrogenase

-is inhibited by ATP and NADH and is activated by ADP and NAD⁺

ɑ-ketoglutarate dehydrogenase complex

-is inhibited by ATP and succinyl-CoA

catabolic pathways

-break down larger molecules into smaller ones

-feed into the citric acid cycle

while many catabolic processes occur in the cytoplasm…

-their end products are permeable to the mitochondrial membrane and can participate in the citric acid cycle

-the breakdown of carbohydrates, lipids and amino acids can all result in metabolic products that can supply the citric acid cycle intermediates

anabolic pathways

-build up larger molecules from smaller ones

-are fed from the citric acid cycle

while the citric acid cycle occurs in the mitochondria…

-many intermediates are permeable to the mitochondrial membrane and can participate in other pathways

-the building up of carbohydrates, lipids and amino acids can all result from metabolic intermediates that are supplied from the citric acid cycle

catabolic and anabolic pathways

-are governed by the needs of the cell and the nutrients that feed the citric acid cycle will need to be replenished if the cell is to remain viable