Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

Abstract

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...

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

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Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

“…Moreover, a unique K ϩ ion-binding pocket in the human E1b ␣ subunit is crucial for the conformation of a cofactor-binding loop (7). The E1b ThDP-binding fold has also been observed recently in the pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (10).…”

The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation

“…Detailed structures of individual components of PDCh (31)(32)(33)(34)(35) and PDCec (36 -39) have been determined by x-ray crystallography (Fig. 2).…”