The hallmark of enzyme catalysis is the high affinity of enzymes for their transition states, but the origin of this tight binding is generally not well understood. This application describes experiments to probe the mechanism by which the large intrinsic binding energy of substrate phosphodianion groups is utilized by a variety of enzymes to stabilize the transition state for formation of an unstable carbanion intermediate. Our central hypothesis is that flexible "phosphate gripper" loops are wide-spread conserved protein elements that provide binding energy that may be utilized for stabilization of the transition state for formation of an enzyme-bound carbanion. The enzymatic reactions of interest include aldose-ketose isomerization, sugar epimerization, aldol condensation and decarboxylation. We will use our "two-part substrate" protocol, where the substrate phosphodianion group, modeled by exogenous phosphite dianion, is detached from the portion of the substrate that undergoes chemical reaction. The specific transition state stabilization from phosphodianion binding interactions can then be quantified from the observed activation of the enzyme by exogenous phosphite dianion towards catalysis of the reaction of the second substrate fragment. Four projects are described to examine both the generality and the mechanism of transition state stabilization arising from flexible loop-phosphite interactions. (1) We will examine members of the orotidine 5'-monophosphate decarboxylase (OMPDC) superfamily. The goal is to determine whether the structurally conserved phosphate gripper loops of these enzymes share the common function of providing specific stabilization of carbanion intermediates. (2) We will examine the relationship between the length of the phosphate gripper loop and the utilization of enzyme-phosphodianion binding interactions for two enzymes that catalyze epimerization of phosphorylated sugars differing in length by only one carbon atom. (3) We will probe the mechanism by which OMPDC achieves its enormous 1017-fold rate acceleration for the chemically difficult decarboxylation of orotidine 5'-monophosphate. We will probe the mechanism by which interactions between the enzyme and the substrate phosphodianion are utilized in stabilization of the transition state for decarboxylation at the distant pyrimidine ring, and address other questions about the enigmatic mechanism of action of this enzyme. (4) We will continue our studies of the role of flexible loop-phosphodianion interactions in stabilization of the transition state for formation of the enediol(ate) intermediate of the aldose-ketose isomerization of triose phosphates catalyzed by triose phosphate isomerase. A major goal is to provide a full description of the physical mechanism by which the movement of flexible catalytic loops acts as a "switch" to turn on stabilizing transition state interactions.
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 nonenzymatic reactions may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related issues. The focus of this application is the critical role of flexible phosphate gripper loops in enzymatic catalysis and the results may spur efforts to develop novel enzyme inhibitors that specifically target these loops.
|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|
|Tsuji, Yutaka; Richard, John P (2016) Formation and Mechanism for Reactions of Ring-Substituted Phenonium Ions in Aqueous Solution. J Phys Org Chem 29:557-564|
|Richard, John P; Amyes, Tina L; Malabanan, M Merced et al. (2016) Structure-Function Studies of Hydrophobic Residues That Clamp a Basic Glutamate Side Chain during Catalysis by Triosephosphate Isomerase. Biochemistry 55:3036-47|
|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 :|
|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|
|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|
|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|
|Tsuji, Yutaka; Richard, John P (2015) Swain-Scott Relationships for Nucleophile Addition to Ring-Substituted Phenonium Ions. Can J Chem 93:428-434|
|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|
|Zhai, Xiang; Malabanan, M Merced; Amyes, Tina L et al. (2014) Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion. J Phys Org Chem 27:269-276|
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