Model studies in water are proposed to characterize the kinetic reactivity, and equilibrium stability of highly unstable tertiary carbocation, oxocarbonium ion and thiol ester enolate intermediates of enzyme-catalyzed 1,3 allylic rearrangements, glycosyl transfer, and Claisen condensation reactions, respectively. Although these species have long been putative enzymatic reaction intermediates, direct evidence for their formation is lacking, and there are many unanswered questions about the nature of their existence both in solution and at an enzyme active site. The following problems are addressed in this proposal. 1) Substitution reactions at t-butyl and ring-substituted cumyl derivatives will be studied in order to: a. Determine the lifetimes for carbocations that form as reaction intermediates; b. Determine the importance of concerted bimolecular substitution reactions at tertiary carbon; and c. Characterize the solution reactivity of tertiary carbocations in preparation for studies on isopentenyl pyrophosphate isomerase. 2) Experiments will be performed to determine if there is a simple relationship between oxocarbonium ion lifetime, and the occurrence of general acid- base catalysis of otocarbonium ion formation and breakdown. These model studies will define the conditions under which general acid-base catalysis contributes to enzymatic catalysis of glycosyl cleavage. 3) The rate constants for the formation and breakdown of a thiol ester enolate will be determined for buffer- and solvent-catalyzed reactions, and combined to give the pKa for carbon deprotonation. The enolate lifetime in the presence of buffer acids will be estimated in order to establish whether or not these carbanions are stable enough to exist as intermediates of enzyme catalyzed claisen condensation reactions. The (14C)- labelled thiol ester enolate will be generated in the presence of an enzyme such as thiolase and its cosubstrate in order to determine if the enolate is kinetically competent to serve as a reaction intermediate. It has been demonstrated numerous times the fundamental studies on enzyme mechanisms may play a deep- seated role in solving health related problems.
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