This competitive renewal application seeks continued support for our major research effort directed toward the elucidation of fundamental new chemistry of enzyme and inhibitor action. During the next funding period, we will continue our work in three major areas: studies on the activation and chemical mechanism of E. coli pyruvate formate-lyase, further investigations on the mechanism of halide eliminations through intervening aromatic rings by enzymes which catalyze carbanionic mechanisms and some final studies on the stereospecificity of glyoxalase I catalysis. Our effort will focus on the key enzyme of prokaryotic anaerobic glycolysis, pyruvate formate-lyase (PFL). Recent work, including our own, has provided suggestive evidence that the reaction may proceed by an enzymatically unprecedented homolytic cleavage of pyruvate. Our approach will involve a thorough study of the mode inactivation of PFL by hypophosphite, acetylphosphinate and propargylic acid. The latter two were recently discovered by us to be extremely potent inactivators of the enzyme. The novel mode of activation of PFL will also be investigated. These studies will require a broad effort with expertise in synthesis, enzyme purification, stable and radio isotope analyses, high field NMR and EPR studies. We have recently demonstrated that novel quinodimethane intermediates are generated from p-halomethyl-substituted aromatic substrates by enzymes which proceed by putative carbanionic mechanisms. The phenomenon has been established for glyoxalase I, benzoyl-formate decarboxylase and mandelate racemase. These studies will now be extended to the flavin-dependent D- amino acid oxidase and, in an attempt to determine if radical mechanisms also can catalyze these eliminations, to monoamine oxidase. This work is of particular significance to the area of drug design and enzyme inactivation by quinone methide intermediates. Finally, we will execute some final experiments to definitely establish that glyoxalase I has the unique and evolutionarily significant ability to catalyze a non-stereo-specific proton abstraction from one carbon followed by a completely stereospecific protonation at an adjacent carbon by single active site base. We believe that these studies are relevant to a number of basic issues in biochemistry and medicine and that new chemistry and enzymology will result from this work.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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University of Maryland College Park
Schools of Earth Sciences/Natur
College Park
United States
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Parast, C V; Wong, K K; Lewisch, S A et al. (1995) Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419. Biochemistry 34:2393-9
Parast, C V; Wong, K K; Kozarich, J W et al. (1995) Electron paramagnetic resonance evidence for a cysteine-based radical in pyruvate formate-lyase inactivated with mercaptopyruvate. Biochemistry 34:5712-7
Landro, J A; Gerlt, J A; Kozarich, J W et al. (1994) The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate. Biochemistry 33:635-43
Wong, K K; Murray, B W; Lewisch, S A et al. (1993) Molecular properties of pyruvate formate-lyase activating enzyme. Biochemistry 32:14102-10
Landro, J A; Brush, E J; Kozarich, J W (1992) Isomerization of (R)- and (S)-glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site. Biochemistry 31:6069-77
Kozarich, J W (1988) S-adenosylmethionine-dependent enzyme activation. Biofactors 1:123-8
Brush, E J; Lipsett, K A; Kozarich, J W (1988) Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage. Biochemistry 27:2217-22
Reynolds, L J; Garcia, G A; Kozarich, J W et al. (1988) Differential reactivity in the processing of [p-(halomethyl)benzoyl] formates by benzoylformate decarboxylase, a thiamin pyrophosphate dependent enzyme. Biochemistry 27:5530-8