Model studies, in water, to characterize the kinetic and equilibrium stability, and mechanisms for the formation and reaction of putative carbanion, carbocation and quinone methide intermediates of enzyme-catalyzed reactions are proposed. (1) It has been suggested that the efficient enzymatic catalysis of deprotonation of alpha-carbonyl compounds results in part from a reduction in the Marcus intrinsic barrier by protonation of the carbonyl oxygen of the bound substrate by an acidic amino acid. However, the origin of the intrinsic kinetic barriers to these reactions is poorly understood, and there are very large uncertalnties in their magnitude. We propose to determine the effect of O-methylation (which models O-protonation) of ring- substituted acetophenones on the intrinsic kinetic barrier to deprotonation of these ketones by carboxylate ions. This will provide a critical test of literature proposals concerning the origin of the intrinsic barrier to proton transfer at carbon, and of the extent to which it can be lowered by interactions with an enzyme. (2) The advantage to concerted acid/base catalysis of reactions in which the product(s) are stabilized by a strong hydrogen bond is not well characterized. We propose to determine the effect of formation of a strong intramolecular hydrogen bond on the stability of the transition state for cleavage of ring- substituted 1-arylethyl salicyl ethers, and the changes in transition-state stabilization with changing substrate structure. The results will define the requirements for optimal enzymatic catalysis by hydrogen bonding. (3) The efficiency of metal ion catalysis of the deprotonation of alpha-carbonyl compounds is not well understood. We propose to characterize the effect of metal dications on the hydroxide-ion-catalyzed abstraction of the alpha- protons of simple carboxylate anions that form strong chelates with the metal dication. (4) The rate laws 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-base catalysis. The advances in the understanding of enzymatic reaction mechanism that result from model studies of nonenzymatic reactions in water may prove critical to drug design (enzyme inhibitors), to the understanding of metabolic pathways and diseases, and to the resolution of other health-related problems.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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State University of New York at Buffalo
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Zhai, Xiang; Reinhardt, Christopher J; Malabanan, M Merced et al. (2018) Enzyme Architecture: Amino Acid Side-Chains That Function To Optimize the Basicity of the Active Site Glutamate of Triosephosphate Isomerase. J Am Chem Soc 140:8277-8286
Reyes, Archie C; Amyes, Tina L; Richard, John P (2018) Primary Deuterium Kinetic Isotope Effects: A Probe for the Origin of the Rate Acceleration for Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase. Biochemistry 57:4338-4348
Richard, John P; Amyes, Tina L; Reyes, Archie C (2018) Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis. Acc Chem Res 51:960-969
Kulkarni, Yashraj S; Liao, Qinghua; Byléhn, Fabian et al. (2018) Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase. J Am Chem Soc 140:3854-3857
Amyes, T L; Malabanan, M M; Zhai, X et al. (2017) Enzyme activation through the utilization of intrinsic dianion binding energy. Protein Eng Des Sel 30:157-165
Amyes, Tina L; Richard, John P (2017) Substituent Effects on Carbon Acidity in Aqueous Solution and at Enzyme Active Sites. Synlett 28:2407-2421
Amyes, Tina L; Richard, John P (2017) Primary Deuterium Kinetic Isotope Effects From Product Yields: Rationale, Implementation, and Interpretation. Methods Enzymol 596:163-177
Reyes, Archie C; Amyes, Tina L; Richard, John P (2017) Enzyme Architecture: Erection of Active Orotidine 5'-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes. J Am Chem Soc 139:16048-16051
Reyes, Archie C; Amyes, Tina L; Richard, John P (2017) A reevaluation of the origin of the rate acceleration for enzyme-catalyzed hydride transfer. Org Biomol Chem 15:8856-8866
Reyes, Archie C; Amyes, Tina L; Richard, John P (2016) Structure-Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase. J Am Chem Soc 138:14526-14529

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