REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

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Model studies in water to characterize the kinetic reactivity, equilibrium stability and mechanisms for the formation and reaction of putative carbanion, carbocation and quinone methide intermediates of enzyme-catalyzed reactions are proposed. The following problems will be studied: (1) Oxygen and thiol ester enolates have been proposed as intermediates for a wide range of enzymatic reactions. However, there is scant direct evidence for their formation in water or at enzyme active sites. We will generate the putative enolate intermediates of seven enzymatic reactions by general base-catalyzed deprotonation of the respective carbonyl precursors, and determine the relative thermodynamic stabilities of these enolates. (2) The relative advantage for metal ion catalysis of deprotonation at carbon is not known. This will be determined by a comparison of metal-ion activation of proton transfer with the activation provided by a CH3 + group. (3) A series of alpha-substituted enol phosphotriesters will be synthesized and their potential use as precursors to alpha-substituted enols and ketenes, or as suicide enzyme inhibitors will be explored. (4) The enzymatic source of methylglyoxal in mammalian tissues will be purified and characterized in order to determine whether it is identical with triosephosphate isomerase. This work may resolve the enigma of the metabolic role of glyoxalase I and II. (5) It is not known if the reaction catalyzed by isopentenyl pyrophosphate isomerase proceeds through a tertiary carbocation reaction intermediate or if it proceeds through a highly polar, carbocation-like, transition state of a concerted reaction mechanism. We will determine whether a model nonenzymatic 1,3-allylic isomerization reaction follows a stepwise or a concerted mechanism. (6) The rate law for the reactions of a simple quinone methide with nucleophilic reagents will be characterized in order to determine if these reactions are subject to general acid and/or general base catalysis. The results of this work will be compared with results of studies on nucleophilic addition reactions at the carbonyl group. (7) Experiments will be performed to determine whether there is a simple relationship between carbocation lifetime and the occurrence of general acid-base catalysis of oxocarbenium ion formation and breakdown. Advances in the understanding of enzyme mechanisms that result from model studies of nonenzymatic reactions may prove critical for drug design (enzyme inhibitors), to the understanding of metabolic pathways and diseases, and to the resolution of other health-related problems.