Enzymes are distinguished from small molecule catalysts by their highly evolved reaction mechanisms that enable the utilization of binding interactions with non-reacting portions of the substrate for transition state stabilization. Innovative protocols developed at Buffalo will be used to test the hypothesis that specificity in transition state binding is obtained by utilization of the intrinsic binding energy of substrate fragments - such as a phosphodianion, pyrophosphotrianion, or ribofuranosyl ring - to drive energetically demanding and structurally complex changes from an inactive open enzyme to a catalytically active caged Michaelis complex with substrate. The relationship between the extraordinary 1017-fold rate acceleration for decarboxylation of orotidine 5'-monophosphate (OMP) catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) and the extensive movements of a phosphodianion gripper loop and a pyrimidine umbrella that accompany formation of the Michaelis complex will be examined. (1) The effects of multiple mutations at both the gripper loop and the pyrimidine umbrella will be examined, to determine whether enzyme activation is the result of cooperative closure of these two protein structural elements. (2) Activation of OMPDC toward catalysis of decarboxylation of 5-fluoroorotate, the ultimate truncated substrate, by exogenous cis-tetrahydrofuran-3,4-diol and phosphite dianion will be examined. (3) The activating nature of the interactions between OMPDC and the ribofuranosyl hydroxyl groups of OMP will be probed in mutagenesis experiments and in studies of substrate analogs lacking these hydroxyl groups. The effect of site-directed mutations on dianion activation of the reduction of the truncated substrate glycolaldehyde by NADH catalyzed by glycerol 3-phosphate dehydrogenase (GPDH) will be determined. The data will be compared with those from published studies of OMPDC and triosephosphate isomerase (TIM), in order to define the essential features of the active site architectures of TIM, OMPDC and GPDH that enable dianion activation of reactions proceeding through chemically diverse transition states. The temperature dependence of the primary deuterium kinetic isotope effect on the phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde by NADH/NADD will be examined. It will be determined whether these isotope effects are consistent with a classical model for hydride transfer or with a more complex model involving quantum mechanical tunneling through the barrier. The kinetic parameters for isomerization of isopentenyl monophosphate and for incorporation of deuterium from solvent D2O into the truncated neutral substrate 2-methylpropene catalyzed by isopentenyl diphosphate isomerase (IDI) will be determined. Activation of IDI-catalyzed deuterium exchange into the truncated substrate by the isohypophosphate trianion substrate piece will be examined, in order to test the proposal that binding interactions between IDI and the substrate pyrophosphotrianion group are utilized to stabilize the transition state for formation of an enzyme- bound tertiary carbocation.

Public Health Relevance

Enzyme catalysts are one of the principal components of all living systems and there are many diseases that arise from the malfunction or deficiency of only a single enzyme. Advances in the understanding of enzyme catalysis from mechanistic studies of enzymes and of the corresponding nonenzymatic reactions in water may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related issues. This proposal describes fundamental studies of the origins of the high specificity of enzyme catalysts for binding of their reaction transition states, the results wll be of particular relevance in guiding the design of tight-binding transition state analog inhibitor of therapeutic interest.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Barski, Oleg
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State University of New York at Buffalo
Schools of Arts and Sciences
United States
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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
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
He, Rui; Reyes, Archie C; Amyes, Tina L et al. (2018) Enzyme Architecture: The Role of a Flexible Loop in Activation of Glycerol-3-phosphate Dehydrogenase for Catalysis of Hydride Transfer. Biochemistry 57:3227-3236
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
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

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