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Author Finnegan, Steffan ♦ Agniswamy, Johnson ♦ Weber, Irene T.
Sponsorship USDOE
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword BASIC BIOLOGICAL SCIENCES ♦ GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE ♦ ALANINES ♦ ATOMS ♦ CATALYSIS ♦ CHAINS ♦ CHOLINE ♦ CRYSTALLOGRAPHY ♦ ELECTROSTATICS ♦ ENZYMES ♦ HYDROGEN PEROXIDE ♦ ISOALLOXAZINES ♦ KINETICS ♦ MUTAGENESIS ♦ OXIDASES ♦ OXIDATION ♦ OXYGEN ♦ SUBSTRATES ♦ THREONINE ♦ VALINE ♦ WATER
Abstract The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants {ge} 10{sup 5} M{sup -1} s{sup -1} and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 {angstrom} from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.
ISSN 00062960
Educational Use Research
Learning Resource Type Article
Publisher Date 2010-11-03
Publisher Place United States
Journal Biochemistry-US
Volume Number 49
Issue Number 13
Organization Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)


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