Lecture 12: Citric acid cycle 1

What are the 2 ways of generating pyruvate

Glycolysis (major source)

Breakdown of amino acids like alanine, cysteine, glycine, serine, threonine and tryptophan

What needs to happen to the pyruvate before it is removed

Needs to be processed otherwise it can cause disturbances in the metabolic pathways

Under anaerobic conditions what is pyruvate converted to in mammals and plants/ microorganisms

Lactate in mammals and ethanol in plants

What are the 2 reactions it can undergo under aerobic conditions

Can be oxidised to CO2 (catabolism) - which is eventually used to generate ATP

or used for biosynthesis (anabolism) - i.e. synthesis of glucose from pyruvate in gluconeogenesis

What are the products of glycolysis

2ATP (2 must be reused to activate glucose)

2 pyruvate

2 NADH

What then needs to happen for glycolysis to continue

Pyruvate has to be removed and NADH has to be converted back to NAD+

Under anaerobic mammalian conditions, what enzyme is used to reduce pyruvate, what is the exact product (S/D)

Lactate dehydrogenase

Forms NAD+ and S-Lactate

How does the structure of lactate differ to that of pyruvate, is the reaction reversible or non-reversible

C=O (ketone group) reduced to a hydroxyl group, reversible reaction

What does pyruvate first need to be converted to in order to produce ethanol

Ethanal

What enzyme does this, what are the 2 coenzymes/cofactors needed

Pyruvate decarboxylase, TPP (thiamine pyrophosphate) and Mg2+

What does this first reaction result in

Decarboxylation of pyruvate, releasing carbonyl group as CO2 (3→ 2 carbon molecule)

What is the enzyme used to convert ethanal to ethanol, what else is needed

Alcohol dehydrogenase, NADH + H+ has to be converted to NAD+

What part of this reaction is reversible/non-reversible

Decarboxylation is non-reversible and reduction of ethanal is reversible

Where does the aerobic catabolism of pyruvate occur

In the mitochondrial matrix

How does pyruvate enter the matrix

Facilitated diffusion through the outer membrane and through a specific transporter protein through the inner membrane

What is the first step of the aerobic catabolism of pyruvate

Pyruvate converted to acetyl CoA

What aspect of the mitochondrial matrix makes it suited for aerobic catabolism

Contains a large mixture of enzymes requires for oxidation of pyruvate to acetyl coA

What is the overall reaction from pyruvate to acetyl-CoA, what are the 5 (co)enzymes and cofactors used

Pyruvate oxidised to acetyl-CoA using a pyruvate dehydrogenase complex, CoA-SH, TPP, lipoamide FAD AND the conversion of NAD+ to NADH + H+

This releases CO2 and forms acety-CoA

Is this stage reversible or not

Irreversible due to loss of CO2

What is the pyruvate dehydrogenase complex

Large multi enzyme complex which performs the reaction

What are the 3 core enzymes in the PDC and their E number

Pyruvate dehydrogenase (E1)

Dihydrolipol transferase (E2)

Dihydrolipoyl dehydrogenase (E3)

What does pyruvate dehydrogenase do, what is it’s cofactor

Performs decarboxylation using its TPP cofactor

What does dihydrolipol transferase do, what is it’s coenzyme

Synthesises acetyl-CoA, using Lipoate and CoA

What does dihydrolipoyl dehydrogenase do, what coenzymes does this use

regenerates oxidised lipoamide which allows the cycle to continue - using FAD and NAD+

What’s special about E3 regenerating lipoamide

Lipoamide is the cofactor used by E2

What proportion of E1:E2:E3 do mammals have in the complex

30 E1, 12 E2 and 12 E3

How many coenzymes and cosubstrates does the PDC contain - why are these needed

5, they enable the complex to convert pyruvate to acetyl-CoA

What are the full names of these 5 coenzymes/cosubstrates

Thiamine pyrophospate (TPP) - which also needs Mg2+

Lipoamide

Coenzyme A

Flavin adenine dinucleotide (FAD)

Nicotinamide adenine dinucleotide (NAD)

FAD and NAD both in oxidised forms to start

What is the first step of the pyruvate dehydrogenase complex

Pyruvate binds to E1

TPP on E1 attacks the carbonyl carbon of the pyruvate which releases CO2

The remaining fragment bound to the TPP is then oxidised to an acetyl group

When this happens, where are the electrons transferred to

The disulfide bond of lipoate on the E2 (dihydrolipoyl transacylase) complex which is catalysed by E1

What are the products of this

Acetyl-dihydrolipoamide

TPP is regenerated

How is the acetyl group transferred to coenzyme A

CoA-SH attacks the acetyl-lipoamide

The acetyl group is transferred to CoA from lipoamide

How does this occur

Exchanging the sulfur in the lipoamide for the sulfur in coenzyme A (forms CoA-SH) - which is CoA in its reduced form)

What does this produce

Acetyl-CoA

Reduced dihydrolipoamide

What does the reduced lipoamide transfer electrons to

FAD

Lipoamide is restored to its oxidised disulfide form

FAD turns into FADH2

What enzyme catalyses this

E3

How does NADH + H+ get formed

The FADH2 transfers electrons to NAD+, regenerating FAD

What is the overall PDHC reaction

Pyruvate + CoA + NAD+ → Acetyl-CoA + CO2 + NADH

What is the bond between the intermediates and the compounds

Covalent bonds

What adaptation of lipoamide means it can move into three different active sites

It is a very flexible molecule

What does substrate channelling allow

Allows steps to proceed at a rate not limited by the free concentration of substrate (can occur faster than that limited by diffusion), which prevents side reactions