This grant seeks continued support for our research on the mechanism of enzyme action. Specifically, we propose to investigate three enzymes which are involved in the metabolism of three-carbon units. Our work on glyoxalase I will focus on the identification of the active site base responsible for proton transfer using some of the principles of our recent finding of """"""""mirror-image"""""""" catalysis by glyoxalase I. In addition, other studies are designed to probe the nature of the partitioning reaction of halomethylglyoxals by the enzyme. We will attempt to determine the contributions of product release from the enzyme and halogen orientation on the partitioning reaction. Our studies on lactate racemse will focus on two major issues. First, we will investigate the question of proton versus hydride transfer in the racemization step. We will accomplish this using many of the techniques that we have established in the glyoxalase I studies. Second, we will address the question of substrate activation in this system. There has been some speculation that lactate racemase may involve a covalent enzyme-substrate complex or a """"""""charged"""""""" enzyme. Extreme difficulty in the purification and stabilization of this enzyme has prevented the resolution of this issue. We propose to develop a general method for the detection of a """"""""charged"""""""" enzyme which we call anhydride detection by intermolecular oxygen scrambling. The technique relies on the implied radomization of carboxylate oxygens in an acyl exchange mechanism. Our technique will involve a novel utilization of 18O perturbations on 13C NMR resonances. This method will be extended to other enzymes where """"""""charged"""""""" forms and anhydride intermediates have been proposed. Finally, we would like to initiate studies on elucidating the molecular mechanism of pyruvate-formate lyase, an enzyme which bears some similarity to the two discussed above in terms of ambiguity of electron flow. We propose studies designed to determine the direction of bond cleavage, the intermediates formed on the enzyme, and the function of enzyme activation by SAM, flavodoxin, and Fe(II)-protein complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035066-05
Application #
3287112
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-09-01
Project End
1989-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Maryland College Park
Department
Type
Earth Sciences/Resources
DUNS #
City
College Park
State
MD
Country
United States
Zip Code
20742
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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