Bioc 203: PDC and Krebs Cycle

PDC

Pyruvate Dehydrogenase Complex

• pyruvate gets oxidized

Conversion of Pyruvate to acetyl-CoA

• done by PDC (multi-enzyme complex)

• occurs in the mitochondrial matrix

• acetyl-coA is the fuel of the krebs cycle

• irreversible at physiological conditions

• involves a decarboxylation/oxidation of pyruvate to acetate in the form of a thioester, followed by the formation of acetyl CoA

Pyruvate + NAD⁺ + Co^AA-SH —> Acetyl-CoA + CO₂ + NADH + H⁺

4º Structure of PDC

Complex is composed of many copies of the enzymes and cofactors

Enzymes:

• E1: Pyruvate dehydrogenase

• E2: Dihydrolipoyl transacetylase

• E3: Dihydrolipoyl dehydrogenase

Cofactors:

• thiamine pyrophosphate (TPP), bound to E1

• lipoamide, bound to E2

• NAD⁺, free

• FAD, bound to E3

• CoAsh, free

CoEnzyme A and Acetyl CoA

Coenzyme A: aka CoA or CoAsh

• carrier of acyl groups

• forms high energy thioester bonds

Acetyl CoA + H₂O ⇌ Acetate + CoASH

• ∆Gº’ = -31 KJ/mol

Thiamine Pyrophosphate (TPP) and hydroxyethyl TPP

• derived from vitamin B₁

• forms a reactive carbanion

• carries aldehyde

• usually causes a decarboxylation

Lipoamide and Lipoic acid

• lipoic acid is attached to a lysine R group, forming lipoamide

  • lipoamide is very flexible

• oxidize aldehydes to acyl groups, resulting in the acyl group being bound via the disulfide group

  • acting as a robotic arm

Mechanism of PDC

1. Pyruvate enters E1, binds to TPP and is decarboxylated to form the intermediate hydroxyethel, TPP

2. the oxidized limpoamide arm enters E1

3. the hydroxyethyl group is oxidized to an acetyl group, and is bound to the lipoamide arm. The arm is now reduced to a dihydrolipoyl group

4. the reduced arm carrying the acetyl group moves into E2 where the acetyl group is transferred to CoASH, forming acetyl CoA. Acetyl-CoA leaves the enzyme

5. the reduced lipoamide arms moves into E3, where it is reoxidized. In the process, FAD is reduced FADH₂

6. NAD⁺ enters E3 and reoxidizes FADH₂ back to FAD. NAD⁺ is reduced to NADH and H⁺ which leaves the enzyme

Back to step 1

Mechanism of PDC: Figure

Regulation of the PDC

• high conc. of acetyl CoA allosterically inhibits E2

• high [NADH] allosterically inhibits E3

• The main control is on E1, where phosphorylation by a kinase leads to inhibition of E1 and thus the complex

  • this kinase is the PDC associated kinase (PDCAK)

• acetyl CoA, NADH and ATP all stimulate the kinase

• pyruvate, NAD⁺ and ADP all inhibit the kinase

• general phosphatases will gradually remove the phosphate from E1, allowing E1 to reset and become active

• Cell signaling, such has high [Ca²⁺] and insulin activates the PDC associated phosphatase (PDCAP) that results in the rapid dephosphorylation of E1

Regulation od PDC: Figure

Krebs Cycle

• metabolic hub of the cell

• completely oxidizes acetyl CoA to CO₂ , and in the process, generates high energy e^- in the forms of NADH and FADH₂ and GTP

• source of many biological precursors

• occurs in the mitochondrial matrix

Reaction 1: Synthesis of Citrate from Acetyl CoA and Oxaloacetate (Citrate synthase)

• citric synthase forms citrate by binding oxaloacetate to acetyl coA

  • going from C4 to C2

• 2 part process:

  • aldol condensation to form citryl CoA

  • hydrolysis of citryl CoA to citrate and CoASH (very favorable step)

Synthase vs Synthethase

Synthase: an enzyme catalyzing a synthetic reaction in which 2 units are joined without the direct participation of ATP (NTP)

Synthethase: same as above, but ATP (NTP) is directly required

Reaction 2: Conversion of Citrate to Isocitate (Aconitase)

• aconitase converts citrate to isocitrate

  • moving the OH group

  • dehydration reaction to form cis-aconitate

  • followed by a hydration step to generate iso-citrate

  • ∆Gº’ > 0, but the reaction is driven by both reaction 1 and 3

Reaction 3: Decarboxylation/Oxidation of Isocitrate to α-ketoglutarate (isocitrate dehydrogenase)

• irreversible reaction

• isocitrate is oxidized/decarboxylated to α-ketoglutarate, NADH and CO₂ are produced

• 2 step process:

  • isocitrate is oxidized to ozalosuccinate, generating NADH

  • oxalosuccinate is decarboxylated to α-ketoglutarate spontaneously (5 carbons)

• Note: the CO₂ lost did not originate from the acetyl CoA that just entered the cycle

Reaction 4: Oxidation/Decarboxylation of ⍺-ketoglutarate to succinyl-CoA (⍺-ketoglutarate dehydrogenase complex)

• irreversible reaction

• ⍺-ketoglutarate is decarboxylated/oxidized and bound to CoASH by ⍺-ketoglutarate dehydrogenase complex. This generates succinyl CoA, NADH and CO₂

• occurs by the same method as the PDC

  • same cofactors, similar E1 and E2 enzymes and identical E3 enzyme

• back to 4 carbons

Conserved Mechanism for Oxidative Decarboxylation

Reaction 5: Conversion of Succinyl CoA to Succinate with GTP generation (Succinyl CoA synthetase)

• succinyl CoA synthetase converts succinyl CoA to succinate, generated GTP and CoASH

  • the reaction is driven by the negative ∆Gº’ in the cleavage of thioester bond

• GTP can be converted to ATP by nucleoside diphosphate kinase

  • GTP +ATP⇌GDP + ATP

• There are isoforms of succinyl CoA synthethase that use ADP instead of GDP

• the next steps are involved in the regeneration of oxaloacetate

Reaction 6: Oxidation of Succinate to Fumarate (Succinate Dehydrogenase)

• succinate dehydrogenase oxidizes succinate, generating FADH₂ and fumarate (trans)

• free energy change is not high enough to reduce NAD⁺

• succinate dehydrogenase is part of complex 2

Reaction 7: Adding water across the double bond of fumarate to form malate (fumarase)

Fumarase adds water across the double bond, forming L-Malate

Reaction 8: Oxidation of Malate to Oxaloacetate (malate dehydrogenase)

• malate dehydrogenase oxidizes malate to oxaloacetate, generating NADH

• cyclic is complete

Overall (1 turn of Krebs Cycle)

Regulation of the Krebs Cycle

Allosteric regulation

1. Isocitrate dehydrogenase

   1. stimulate by ADP

   2. inhibited by NADH and ATP

2. ⍺-ketoglutarate dehydrogenase complex

   1. ATP, NADH and succinyl CoA all inhibit the enzyme

Optional (only in bacteria)

3. Citrate synthase

   1. inhibited by ATP