2-OXO-3-ALKYNOIC ACIDS, UNIVERSAL MECHANISM-BASED INACTIVATORS OF THIAMIN DIPHOSPHATE-DEPENDENT DECARBOXYLASES - SYNTHESIS AND EVIDENCE FORPOTENT INACTIVATION OF THE PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl sub stituent at carbon 4 was reported by the authors as potent irreversibl e and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) d ependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (199 4) J. Org. Chem. 59, 5763-5766]. The method has been successfully exte nded to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on t hree ThDP-dependent pyruvate decarboxylases: the Escherichia colt pyru vate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-depende nt) component, pyruvate oxidase (POX, phosphorylating; from Lactobacil lus plantarum), and pyruvate decarboxylase (PDC) from Saccharomyces ce revisiae. All three enzymes were irreversibly inhibited by the new com pounds. The 4-carbon acid is the best substrate-analog inactivator kno wn to date for PDHc, more potent than either fluoropyruvate or bromopy ruvate. The following conclusions were drawn from extensive studies wi th PDHc: (a) The kinetics of inactivation of PDH complexes and of reso lved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent . (b) The 4-carbon acid has a K-i 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgC l2. (d) Pyruvate completely protects E1 and partially protects PDHc fr om inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E 1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cyste ines/E1 monomer. The order of reactivity with the 4-carbon acid was PD Hc > POX > PDC. While the order of reactivity with PDHc and POX was 2- oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.