The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In ␣ 2  2 -heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C ␣ ؊C(؍O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Å resolution. We propose a model for the flip-flop action of this enzyme through a concerted ϳ2-Å shuttlelike motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.The thiamin pyrophosphate (TPP) 1 -dependent enzymes perform a wide range of catalytic functions in the pathways of energy production, including decarboxylation of ␣-keto acids followed by transketolation. The enzymes that have been structurally characterized so far, 2-oxoisovalerate dehydrogenase from Pseudomonas putida (1), human branched-chain ␣-ketoacid dehydrogenase (2), bacterial pyruvate dehydrogenase (3), transketolase (4), pyruvate decarboxylase (5), benzoylformate decarboxylase (6), acetohydroxyacid synthase (7), pyruvate oxidase (8), and pyruvate:ferredoxin oxidoreductase (9), have shown a common mechanism of TPP activation by (i) forming the ionic N-H⅐⅐⅐O Ϫ hydrogen bonding between the N1Ј atom of the aminopyrimidine ring of the coenzyme and an intrinsic ␥-carboxylate group of glutamate and (ii) imposing an "active" V-conformation that brings the N4Ј atom of the aminopyrimidine to the distance required for the intramolecular C-H⅐⅐⅐N hydrogen-bonding with the thiazolium C2 atom (Fig. 1). Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1Ј atom of the aminopyrimidine system is strictly connected with the deprotonation of the 4Ј-amino group in that system and eventually abstraction of the proton from C2 and formation of the reactive 4Ј-amino-C2-carbanion (Fig. 1a) (10). This reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used in the family of TPP-dependent enzymes. Within pyruvate dehydrogenase (E1), the first catalytic component enzyme of pyruvate dehydrogenase complex (PDC), this substrate is pyruvate (S 1 ). The cleavage of the central C ␣ -C(ϭO) bond of this substrate proceeds from induction of the intermediate, 4Ј-imino-2-(2-hydroxypropionyl)thiamin pyrophosphate, i.e. lactyl-TPP (LTPP) (Fig. 1b), followed by conversion to 4Ј-imino-2-(1-hydroxyethyl) thiamin pyrophosphate (HETPP) with release of carbon dioxide (P 1 ) (Fig. 1c). The fate of this active C2-␣-carbanion/enamine HETPP differs among various TPP-dependent enzymes depending on the nature of t...
At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.
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