Isotope Effects on the Urate Oxidase Reaction

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

The chemical mechanisms of enzyme-catalyzed reactions with O2 are of particular interest, both because of the importance of O2 to living organisms, and because reactions between O2 and organic molecules are formally forbidden by the rules of quantum mechanics. In order to facilitate reactions with O2, nature has evolved enzymes that contain cofactors, either organic or inorganic, which readily react with O2. The enzyme urate oxidase is quite unusual in that it requires O2 as a substrate, but neither contains nor requires any cofactor. The research outlined is designed to provide a detailed understanding of how urate oxidase facilitates the reaction between O2 and urate. A small but growing number of enzymes has been recognized to catalyze reactions with O2 in the absence of cofactors. The urate oxidase reaction will be probed by measuring 13C, 18O, and 35S isotope effects on substrate oxidation and on the binding of ligands at the active site. Binding isotope effects will be particularly useful for determining how hydrogen-bonding interactions at the active site influence the reactivity of urate. Kinetic isotope effects will help to define the mechanism that urate oxidase employs to enhance the reactivity of urate towards O2. One can envision several means that could be used in the enzymatic reaction. Model studies have shown that the urate dianion can be oxidized readily, and evidence from kinetic studies suggests that urate oxidase does generate the urate dianion at the active site. Since the product of the urate oxidase reaction contains a tetrahedral carbon at C5 while the substrate is planar, the enzyme could also enhance the reactivity of urate by contorting it away from planarity. The enzyme can also vary hydrogen-bonding and pi-stacking interactions with the ligand, to vary its redox potential. All of these mechanisms are expected to generate heavy atom kinetic isotope effects. Furthermore, if binding does induce deformation of the substrate or introduce unique hydrogen bonds, there should be equilibrium isotope effects on the binding of ligands to urate oxidase. Interpretation of the isotope effect data will be facilitated by comparison with calculated isotope effects. Ab initio calculations will be performed to generate wave functions for urate and urate analogs participating in molecular interactions that are expected to occur at the active site. The model proposed for the urate oxidase reaction draws parallels with flavin and pterin cofactor chemistry. Since urate is a substrate rather than a cofactor, isotope effect methodologies can be applied to urate oxidase in ways that are not practical with flavoenzymes or pterin-dependent enzymes. However, the fundamental insights gained should allow of a greater understanding of the chemistry between O2 and flavin and pterin as well. Broader impacts This project will promote teaching and training of graduate and undergraduate students through hands on research experience. These research experiences will be directed to learning mechanistic enzymology and protein chemistry.