Isotopic Probes of Flavoprotein Oxidase Mechanisms

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Flavoenzymes catalyze a wide variety of metabolically significant reactions. Flavoprotein oxidases are among the simplest of these enzymes, catalyzing the transfer of a hydride equivalent from their substrates to molecular oxygen to form hydrogen peroxide. The research proposed here will develop methods utilizing kinetic isotope effects on steady state and rapid reaction kinetics to examine the mechanisms of flavoprotein oxidases, using D-amino acid oxidase (DAAO) as a model. The PI will determine the pH dependence of the V/K values and of the primary deuterium kinetic isotope effects on the V/K values with D-alanine, D-serine, and glycine; determine if the maximal observed isotope effects are the intrinsic values; measure secondary deuterium kinetic isotope effects in order to determine if carbon-hydrogen bond cleavage and flavin reduction in D-amino acid oxidase are stepwise or concerted; and replace the native FAD with artificial flavins with different redox potentials and determine the intrinsic isotope effects. These methods will then be used to evaluate the results of site-directed mutagenesis of individual amino acid residues in DAAO. They will also be applied to two additional flavoprotein oxidases, tryptophan monooxygenase and alcohol oxidase, to ascertain their general applicability. %%% Flavoproteins are a major class of proteins involved in a wide variety of metabolic processes. Elucidation of the catalytic mechanisms of these enzymes can provide insights into the mechanisms of a number of metabolically significant enzymes, into the mechanisms of more complex flavoproteins, and into general biological mechanisms of electron transfer and oxygen activation. This research will yield information pertaining to the mechanism of flavoprotein oxidases using kinetic isotope effects to examine the role of specific amino acid residues in catalysis. General knowledge about flavoprotein carbon-hydrogen bond cleavage as well as about oxygen activation by flavin and pterin dependent enzymes will also be generated.