Pyruvate Dehydrogenase Complex

Pyruvate dehydrogenase complex

Miklós Csala carbohydrates proteins lipids

glucose aminoacids fatty acids

glycolysis acyl-CoA synthetase

pyruvate acyl-CoA

cytosol resp. chain H+ pyruvate acyl-CoA - H+ e acetyl-CoA H+ - O2 H2O e ADP + Pi citrate - H+ cycle e CO ATP ATP 2 mitochondrium synth. Glycolysis: the net reaction

+ C6H12O6 + 2NAD + 2ADP + 2Pi

- + 2C3H3O3 + 2NADH + 4H +2ATP Mitochondrium outer membrane inner membrane Outer membrane contains pores (composed of porin proteins). These pores are permeable for small molecules so intermembrane space is basically equivalent with the cytosol.

Inner membrane is impermeable and small cytosol molecules need transporter proteins to cross.

intermembrane cristae space matrix Pyruvate carrier

Symport of pyruvate and proton: secondary active transport driven outer by respiration. membrane

inner membrane

pyruvate pyruvate

H+ H+ matrix + cytosol (lower [H ]) (higher [H+]) Oxidative decarboxylation of pyruvate without PDC

+ H -2H oxidation

ionization -H+ CO2 H2O pyruvate acetaldehyde acetaldehyde- acetate hydrate

The overall reaction is spontaneous irreversible. Oxidative decarboxylation of pyruvate by PDC Net reaction:

pyruvate Some free energy is captured if - CH3-CO-COO the reaction is catalyzed by PDC:

+ NAD CoA-SH 1. PDC oxidizes with electron carriers (fuels oxidative phosphorylation) and 2. PDC binds acetic acid to CoA NADH CO2 (creates a high energy

CH3-CO-S-CoA thioester bond). acetyl-CoA Acetaldehyde and acetate are bound to prosthetic groups within the complex. PD complex

•Three metabolic enzymes and two regulatory enzymes in the complex:

E1: pyruvate dehydrogenase (TPP, prosthetic group) E2: dihydrolipoyl transacetylase (lipoic acid, prosthetic group) E3: dihydrolipoyl dehydogenase (FAD, prosthetic group) (two coenzymes involved: NAD+ and CoA-SH) PDH kinase PDH phosphatase.

• Several (12-24) copies of each enzyme with E2 in the center.

• The complex is as big as a ribosome. Prosthetic groups: TPP

Susceptible to acidic dissociation that makes it a good nucleophilic reagent (carbo-anion).

TPP-containing enzymes: pyruvate dehydrogenase (E1 of PDC) α-ketoglutarate dehydrogenase (E1 of αKGDC) branched ketoacid dehydrogenase (E1 of BKADC) transketolase Prosthetic groups: TPP TPP always carries an aldehyde group in aldol linkage

TPP-containing enzymes: pyruvate dehydrogenase (E1 of PDC) α-ketoglutarate dehydrogenase (E1 of αKGDC) branched ketoacid dehydrogenase (E1 of BKADC) transketolase Prosthetic groups: lipoic acid

Disulfide can be reduced to di- thiol, which can form thioester. Forms amide bond with ε- amino group of a Lysine residue

lipoic acid

lipoamide (prosthetic group)

dihydrolipoic acid

Enzymes containing lipoic acid:

E2 (transacylase) in PDC, αKGDC and BKADC complexes Prosthetic groups: lipoic acid E1: pyruvate dehydrogenase

decarboxylation

E2: dihydrolipoyl transacetylase

TPP carboanion

oxidation transfer E2: dihydrolipoyl transacetylase again

transfer E3: dihydrolipoyl dehydrogenase

(re-)oxidation

transfer of electrons onto a mobile carrier

NADH + H+ NAD+ Reaction cycle of PDC

Lysyl lipoamide groups act as extended swinging arms between TPPs and FADs. What happens with NADH and acetyl-CoA?

NADH All NADH produced in the mitochondrial matrix fuels respiration (and oxidative phosphorylation). There is no other reaction that significantly re-oxidizes NADH in this compartment. Hence PDC reaction is coupled to respiration and can only function in aerobic conditions (although it does not use oxygen directly).

acetyl-CoA It can be further oxidized in citrate cycle or used for lipid biosynthesis but it can not be converted to carbohydrates (e.g. glucose) effectively. Unlike other irreversible reactions, PDC step can not be undone. Regulation of PDC

Allosteric regulation:

Increased levels of acetyl-CoA and NADH inhibit E2, E3.

PDC

Pyruvate dehydrogenase (E1) + Dihydrolipoyl transacetylase (E2) - + Dihydrolipoyl dehydrogenase - (E3) Pyruvate + HS-CoA + NAD+ NADH + acetyl-CoA + CO2 Regulation of PDC

Covalent modification: phosphorylation/dephopsphorylation of E1. NAD+, pyruvate ADP, AMP NADH, acetyl-KoA ATP - + ADP

Pyruvate PDC-kinase Pyruvate dehydrogenase dehydrogenase (E1) (E1) P

Dihydrolipoyl PDC-phosphatase Dihydrolipoyl transacetylase transacetylase (E2) + (E2) Dihydrolipoyl Pi H2O Dihydrolipoyl dehydrogenase dehydrogenase (E3) Ca2+ (E3) Active Insulin Inactive Regulation of PDC

[NADH ] [ATP] [acyl  CoA] Activated by low , low , low , [NAD  ] [ADP] [CoA]

insulin and Ca2+ (exercise in muscle)

[NADH ] [ATP] [acyl CoA] Inhibited by high , high , high [NAD  ] [ADP] [CoA] PDC facts

• Irreversible conversion of pyruvate to acetyl-CoA (oxidative decarboxylation loading NAD+ with electrons).

• Converts a carbohydrate precursor to a compound that can be either degraded or used for lipid (fatty acid, cholesterol) and ketone body synthesis.

• In mitochondrial matrix (present in every cell that contains mitochondria).

• The complex can only function in AEROBIC conditions.

• Three metabolic enzymes and two regulatory enzymes in the complex:

E1 and E2 are different (specific to the substrate) but E3 is identical.

α-ketoglutarate DH (citrate cycle)

CoA CO2

NAD+ NADH

α-ketoglutarate succinyl-CoA branched chain α-ketoacid DH (Val, Leu, Ile catabolism)

CoA CO2

NAD+ NADH branched α-ketoacid branched acyl-CoA Metabolic diseases related to PDC

Beriberi

Hypovitaminosis B1 (thiamine) Frequent in Far-East (little thiamine content of rice). in alcoholics (malnutrition). High [pyruvate] and [α-ketoglutarate] in blood. Neurologic and cardiac symptomes. Can be diagnosed by RBC transketolase assay.

Genetic defects of PDC

E1 or E2 defect: lactic acidosis (and high [pyruvate]) severe neurologic defects in newborn (due to affected ATP and lipid synthesis).

E3 defect: the same plus citrate cycle defect and demaged branched chain amino/keto-acid catabolism Citrate cycle

Miklós Csala carbohydrates proteins lipids

glucose aminoacids fatty acids

glycolysis acyl-CoA synthetase

pyruvate acyl-CoA

cytosol resp. chain H+ pyruvate acyl-CoA - H+ e acetyl-CoA H+ - O2 H2O e ADP + Pi citrate - H+ cycle e CO ATP ATP 2 mitochondrium synth. Alternative names and history

Citrate cycle (or citric acid cycle) is also referred to as tricarboxylate cycle (TCA cycle), Szent-Györgyi cycle (by proud Hungarians) Krebs cycle (by non-Hungarians) Szent-Györgyi-Krebs cycle (by modest Hungarians).

In 1935, Szent-Györgyi demonstrated that little amounts (catalytic amounts) of some dicarboxylates (succinate, fumarate, malate or oxaloacetate) acelerate respiration, and showed the following interconversions: succinate → fumarate → malate → oxaloacetate.

In 1936, Martius and Knoop reported these reactions: citrate → cis-aconitate → isocitrate → ketoglutarate → succinate.

In 1937, Krebs found the missing reaction and the cycle was closed.

„Enzymatic conversion of pyruvate + oxaloacetate to citrate and CO2” means PDC reaction and the entry of acetyl-CoA into the cycle. He showed that the cycle is a major pathway for pyruvate oxidation in muscle. Overall citrate cycle Citrate synthase

IRREVERSIBLE

Overall ΔGo’ = -31.5 kJ/mol. Aconitase Isocitrate dehydrogenase

Oxidative decarboxylation IRREVERSIBLE α-Ketoglutarate dehydrogenase complex

Oxidative decarboxylation IRREVERSIBLE

The complex is very similar to PDC and contains the same E3. Succinyl-CoA synthetase

Substrate level phosphorylation

GTP can pass the phosphoryl group to ADP (NDP kinase). Succinate dehydrogenase

Membrane bound flavoprotein.

Part of succinate : CoQ reductase complex (Complex II of respiratory chain). The two electrons are transferred to CoQ by Fe-S proteins of the complex. Succinate dehydrogenase

Malonate competitively inhibits succinate dehydrogenase.

succinate malonate oxalate Fumarase

(Stereospecific hydration: always L-malate is formed.) Malate dehydrogenase

This reaction is not spontaneous in standard conditions: ΔGo’ = +29.7 kJ/mol.

It is pushed forward by high [malate]/[oxaloacetate] and [NAD+]/[NADH] ratios. Overall citrate cycle

Because of the stereochemistry, the two carbon atoms lost as CO2 during phase I are not the same as those introduced as acetyl-CoA.

Strictly: one “acetyl equivalent” is oxidized to two molecules of CO2 in each round. Net reaction of citrate cycle

+ CH3CO-S-CoA + 3NAD + FAD + GDP + Pi + 2H2O

+ 2CO2 + HS-CoA + 3NADH + 2H + FADH2 + GTP Citrate cycle facts

• Irreversible oxidation of acetyl group (of acetyl-CoA) to 2CO2 + (oxidation loading NAD and FAD with electrons).

• In mitochondrial matrix (present in every cell that contains mitochondria).

• NADH and FADH2 generated in the cycle can only be reoxidized by the respiratory chain.

• Hence the cycle can only function in AEROBIC conditions, although it does not use oxygen directly.

• The intermediates of the cycle are neither produced nor consumed during the reaction (they are always regenerated). They can rather be considered as catalytic cofactors.

• The intermediates can be consumed by other (anabolic) processes, so they must be resynthesized (anaplerosis).

Standard and physiological ΔG ΔGo’ ΔG Rxn Enzyme (kJ/mol) (kJ/mol) 1 Citrate synthase -31.5 negative

2 Aconitase ≈ 5 ≈ 0

3 Isocitrate dehydrogenase -21 negative

4 α-Ketoglutarate dehydrogenase complex - 33 negative

5 Succinyl-CoA synthetase -2.1 ≈ 0

6 Succinate dehydrogenase +6 ≈ 0

7 Fumarase -3.4 ≈ 0

8 Malate dehydrogenase +29.7 ≈ 0 Regulation of citrate cycle

The cycle is activated by [NADH ] low , [NAD  ] [ATP] low , [ADP] [succinyl CoA] low [CoA] and Ca2+ (e.g. in muscle contraction) Amphibolic role pyruvate of citrate cycle carboxylase

The catabolic function does not consume (or produce) the intermediates. They are always regenerated and only act as catalysts.

But the intermediates are consumed by several anabolic pathways.

The cycle would stop without intermediates. It is therefore vital to synthesise them and maintain their catalytic level. This is called anaplerosis („pay back”). GLUCOPLASTIC KETOPLASTIC

glicogen glycerolipids ketone bodies glucose other mono- fatty acids sacharides PDC pyruvate acetyl-CoA

glucoplastic cholesterol aminoacids oxaloacetate citrate bile acids steroids

CO2 Energetics of glucose oxidation Substrate level Oxidative Glucose

2ATP 2NADH 5ATP/3ATP

2 Pyruvate 2NADH 5ATP

2 Acetyl-CoA + 2CO2

6NADH 15ATP 2ATP

2FADH2 3ATP

4ATP 6CO2 28ATP/26ATP Total: 32ATP/30ATP