Patologia e fisiopatologia generale - Pyruvate Dehydrogenase

Derivazione e funzioni di FAD e TPP

O-FAD (Flavin Adenine Dinucleotide) è derivato dalla vitamina riboflavina. Il sistema ad anello dimetilisoalloxazina subisce ossidazione/riduzione. FAD è un gruppo prostetico, permanentemente parte dell'enzima E.

Reazione: 2 OOH C −3 CH O P O P OCH 22 −−+ OOCH N2N SC +acidic HHH NC3 NH2 thiamine pyrophosphate (TPP)

Thiamine pyrophosphate (TPP) è un derivato della tiamina (vitamina B1). La carenza di tiamina porta alla malattia del beriberi. Colpisce soprattutto il cervello, perché TPP è necessario per il metabolismo dei carboidrati e il cervello dipende dal metabolismo del glucosio per l'energia.

OOH C −3 CH O P O P OCH 22 −−+ OOCH N2N SC +acidic HHH NC3 NH2 thiamine pyrophosphate (TPP) meccanismo: −O O+ CH si dissocia facilmente dal C tra N e S nell'anello tiazolico. C O Il carbanione risultante può attaccare il carbonio cheto del piruvato, che è carente di elettroni. piruvato S CH2 CH2 NH acido lipoico lisina OS CH CH CH CH CH C NH (CH ) CH2 2 2

2 2 4lipoamideLipoamide C Oincludes a − +2e + 2Hdithiol thatundergoes HS CH2oxidation/ CH2 NHreduction. OHS CH CH CH CH CH C NH (CH ) CH2 2 2 2 2 4dihydrolipoamide C OS CH2 CH2 NHOS CH lipoic acid lysineCH CH CH CH C NH (CH ) CH2 2 2 2 2 4lipoamide C O− +The carboxyl at the end of lipoic acid's hydrocarbonε-aminochain forms an amide bond to the group of alysine residue of E , yielding lipoamide.2long flexible arm, including hydrocarbon chains ofAlipoate and the lysine R-group, links each dithiol oflipoamide to one of two lipoate-binding domains of E .2S CH2 CH2 NHOS CH lipoic acid lysineCH CH CH CH C NH (CH ) CH2 2 2 2 2 4lipoamide C O− +The lipoate-binding domains are themselves part of aflexible arm of E that extends out from the core of the2multienzyme complex.This allows lipoamide functional groups to swing fromone active site to another within the complex.The E3 binding protein also has bound lipoamide thatcan participate in exchange of reducing

equivalents withlipoamide on E2. H O2HS SR' AsR' As O + HS SR ROrganic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase.These highly toxic compounds react with “vicinal”dithiols such as the functional group of lipoamide.O In the overall reactionHO C CHCoenzyme A-SH + 3 catalyzed by the Pyruvateacetic acid Dehydrogenase complex,the acetic acid generatedO is transferred to+ H OC CHCoenzyme A-S 23 coenzyme A.acetyl-CoA H O OHHC CNH NH2 2The final electron acceptor+ +NAD .is N N− +2e + HR R+NAD NADHThe Pyruvate dehydrogenase complexHydroxyethyl-TPPSequence of reactions catalyzed by PyruvateDehydrogenase complex:pyruvate reacts with the carbanion of1. The keto C ofTPP on E to yield an addition compound.1The electron-pulling (+) charged N of the thiazole ringpromotes CO loss. Hydroxyethyl-TPP remains.22. The hydroxyethyl carbanion on TPP of E reacts with1lipoamide on E . What was the keto Cthe disulfide of 2of

pyruvate is oxidized to a carboxylic acid, as the lipoamide disulfide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl is linked to one of the thiols of the reduced lipoamide as a thioester (~).

Sequence of reactions (continued)

1. Acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA.
2. The reduced lipoamide swings over to the E3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e + 2 H are transferred to a disulfide on E3 (disulfide interchange).
3. The dithiol on E3 is reoxidized as 2 e + 2 H are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.

Acetyl CoA, a product of the Pyruvate Dehydrogenase reaction, is a central compound in metabolism. The "high energy" thioester linkage makes it an excellent donor of the acetate moiety.

glucose-6-P

Glycolysis

pyruvate

fatty acids

acetyl CoA

ketone

bodies

cholesterol

oxaloacetate citrate

Krebs Cycle

Acetyl CoA functions as:

• input to Krebs Cycle, where the acetate moiety is further degraded to CO₂
• donor of acetate for synthesis of fatty acids, ketone bodies, & cholesterol.

Regulation of Pyruvate Dehydrogenase Complex:

• Product inhibition by NADH & acetyl CoA:
• +NADH competes with NAD+ for binding to E3
• Acetyl CoA competes with CoA for binding to E2
• Phosphorylation/dephosphorylation of E1:
• Specific regulatory Kinases and Phosphatases are associated with Pyruvate Dehydrogenase complex within the mitochondrial matrix.
• A family of Pyruvate Dehydrogenase Kinases catalyze E1 phosphorylation, inhibiting the complex.
• These kinases are activated by NADH & acetyl-CoA, providing another way the major products of the Pyruvate Dehydrogenase reaction inhibit the complex.
• Pyruvate Dehydrogenase Kinase activation involves interaction with E1 subunits to sense changes in oxidation state & acetylation of lipoamide caused by NADH & acetyl-CoA.