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Proteins and Enzymes- Emily > TCA Cycle Mechanisms > Flashcards

TCA Cycle Mechanisms Flashcards

1
Q

What reaction is catalysed by citrate synthase?

A

Oxaloacetate + Acetyl-CoA => Citrate

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2
Q

What is the structure of mammalian citrate synthase?

A

Dimer. Each monomer has both a large and a small flexible domain.

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3
Q

What is the mechanism of substrate binding in citrate synthase?

A

Induced fit mechanism. Binding of oxaloacetate induces a large conformational change in the subunits, creating the Acetyl-CoA binding site and shifting the catalytic residues in the active site into their proper orientation.

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4
Q

What type of kinetics is citrate synthase an example of?

A

Sequential ordered kinetics.

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5
Q

What is the mechanism of citrate synthase?

A

Methyl group in Acetyl-CoA is deprotonated by Asp375. Hydrogen bonds stabilise the enolate (citryl-CoA) and His picks up a proton. A negatively charged His nucleophilically attacks the carbonyl to remove CoA and form the citrate.

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6
Q

What reaction is catalysed by aconitase?

A

The isomerisation of citrate to isocitrate, via cis-aconitate.

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7
Q

Why is the isomerisation reaction of citrate to isocitrate needed in the TCA cycle?

A

For decarboxylation to occur, an oxidation reaction must occur to give a C=O group. However in citrate, there is a 3° alcohol which cannot be oxidised. The 3° alcohol is converted to a 2° alcohol during the isomerisation, which can be oxidised.

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8
Q

How is the Fe-S centre attached to aconitase?

A

Ligated by Cys side chains.

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9
Q

What is necessary for the Fe-S centre in aconitase to be active?

A

Ferredoxin (Fe4S4)

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10
Q

What is the role of the Fe-S centre in aconitase?

A

Acts as a Lewis acid. Orders the substrate into the active site.

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11
Q

What is the mechanism of aconitase?

A
  1. E2 anti elimination of the OH and H, this forms water which forms a dative covalent bond with Fe.
  2. Molecule flips, making the C2 position available to the water molecule. Addition of OH.
  3. Anti addition of a hydrogen retained by an Asp residue.
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12
Q

What reaction is catalysed by isocitrate dehydrogenase?

A

The oxidative decarboxylation of isocitrate to form α-KG, via oxalosuccinate (unstable β keto acid).

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13
Q

What are the two stages involved in the isocitrate dehydrogenase mechanism?

A
  1. Base catalysed hydride transfer

2. Keto-enol tautomerisation

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14
Q

What is the isocitrate dehydrogenase mechanism?

A
  1. Base catalysed hydride transfer from isocitrate to NAD.
  2. Decarboxylation of the oxalosuccinate, forming an enolate ion.
  3. Enolate ion is stabilised by a divalent cation.
  4. Keto-enol tautomerisation to form α-KG.
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15
Q

What cofactors are used by isocitrate dehydrogenase?

A

NAD and a divalent cation (Mg or Mn)

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16
Q

What reaction is catalysed by the α-KG dehydrogenase complex?

A

The oxidative decarboxylation of α-KG to form succinyl-CoA.

17
Q

What is the mechanism of α-KG dehydrogenase?

A

Analogous mechanism to pyruvate dehydrogenase. E1 and E2 are analogous enzymes and E3 is identical in both complexes.

18
Q

What reaction is catalysed by succinyl-CoA synthetase?

A

Substrate level phosphorylation of succinyl-CoA to produce succinate and GTP.

19
Q

What is the mechanism of succinyl-CoA synthetase?

A
  1. Orthophosphate displaces CoA to give succinyl-phosphate in an Sn2 reaction.
  2. His residue removes the phosphate to release succinate.
  3. Phosphohistidine undergoes a conformational change.
  4. Phosphate is transferred to GDP (in the nucleotide binding domain) by nucleophilic attack.
20
Q

What reaction is catalysed by succinate dehydrogenase?

A

Stereospecific dehydrogenation of succinate to form fumarate.

21
Q

What is the mechanism of succinate dehydrogenase?

A

E2 elimination mechanism, oxidising CH2 to C=C.

22
Q

What reaction is catalysed by fumarase?

A

The hydration of the C=C bond in fumarate to form S-malate.

23
Q

What is the mechanism of fumarase?

A
  1. General base in active site removes a proton from water.
  2. Nucleophilic attack of the si face of fumarate by OH, forming a carbanion.
  3. Anti addition of a proton from a general acid to the carbanion.
24
Q

How is the carbanion intermediate stabilised in the fumarase mechanism?

A

By the adjacent carboxyl group in malate.

25
Q

What reaction is catalysed by malate dehydrogenase?

A

The dehydrogenation of malate to regenerate oxaloacetate.

26
Q

Where does the equilibrium lie in the malate dehydrogenase reaction?

A

Towards the reactants which is unfavourable. However the reaction is never actually at equilibrium, because OAA is used to make citrate, which drives the malate to OAA conversion as there is always low [OAA].

27
Q

What cofactors are used by succinate dehydrogenase?

A

FAD.

28
Q

What cofactors are used by malate dehydrogenase?

A

NAD.

Proteins and Enzymes- Emily (14 decks)

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