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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29GM039754-01
Application #
3466926
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1988-05-01
Project End
1993-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Reyes, Archie C; Amyes, Tina L; Richard, John P (2016) Enzyme Architecture: A Startling Role for Asn270 in Glycerol 3-Phosphate Dehydrogenase-Catalyzed Hydride Transfer. Biochemistry 55:1429-32
Zhai, Xiang; Amyes, Tina L; Richard, John P (2015) Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase. J Am Chem Soc 137:15185-97
Reyes, Archie C; Koudelka, Astrid P; Amyes, Tina L et al. (2015) Enzyme architecture: optimization of transition state stabilization from a cation-phosphodianion pair. J Am Chem Soc 137:5312-5
Goryanova, Bogdana; Goldman, Lawrence M; Ming, Shonoi et al. (2015) Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase. Biochemistry 54:4555-64
Reyes, Archie C; Zhai, Xiang; Morgan, Kelsey T et al. (2015) The activating oxydianion binding domain for enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation: specificity and enzyme architecture. J Am Chem Soc 137:1372-82
Richard, John P; Zhai, Xiang; Malabanan, M Merced (2014) Reflections on the catalytic power of a TIM-barrel. Bioorg Chem 57:206-12
Richard, John P; Amyes, Tina L; Goryanova, Bogdana et al. (2014) Enzyme architecture: on the importance of being in a protein cage. Curr Opin Chem Biol 21:1-10
Zhai, Xiang; Go, Maybelle K; O'Donoghue, AnnMarie C et al. (2014) Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase. Biochemistry 53:3486-501
Zhai, Xiang; Amyes, Tina L; Richard, John P (2014) Enzyme architecture: remarkably similar transition states for triosephosphate isomerase-catalyzed reactions of the whole substrate and the substrate in pieces. J Am Chem Soc 136:4145-8
Goldman, Lawrence M; Amyes, Tina L (2014) The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations. Anal Biochem 450:20-6

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