The purpose of this project is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. During the coming grant period the emphasis will be on the use of (13)C, (15)N, and (18)0 isotope effects: 1) Isotope effects in the beta-lambda bridge and lambda bridge and lambda-nonbridge oxygens of ATP and several analogs will be used to study the reactions of myosin ATPase and several kinases. (18)0 isotope effects will be used to define the transition state structure of kanamycin nucleotidyl transferase. Triesters containing a cyclic ethylene group and a leaving group with pK 8.6 will permit observation of isotope effects that accompany phosphorane formation and pseudorotation during hydrolysis. 2) With human malic enzyme, (13)C isotope effects will be measured at C-4 of malate or 2-deuteromalate with NAD or acetylpyridine-NAD to pin down the mechanism and rate limiting steps. 3) L-ribulokinase will be crystallized with AMPPNP and either D- or L-ribulose in the active site and the structures determined by X-ray. 4) The compound giving the upfield (31)P NMR signal that may represent carboxy-P in solutions of phosphate in DMF or DMSO containing CO2 will be characterized and tested as a substrate for appropriate enzymes. 5) The mechanism of asparagine synthetase will be studied with (13)C, (15)N and (18)O isotope effects. 6) The mechanism of isocitrate dehydrogenase will be studied with (13)C and deuterium isotope effects to determine the rate limiting steps with alternate substrates and mutant enzymes. 7) The (13)C isotope effect in the =CH2 group of chorismate will be determined to characterize the transition state for the chorismate mutase reaction. 8) (13)C and (15)N isotope effects will be used to study the mechanism of ornithine transcarboxylase. 9) Other collaborations will determine isotope effects on cytidine deaminase, non-enzymatic decarboxylation of hydroxybenzoates, and on selected fiavoprotein-catalyzed reactions.
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