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J. Biol. Chem., Vol. 280, Issue 29, 27103-27110, July 22, 2005

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Role of Active Site Residues and Solvent in Proton Transfer and the Modulation of Flavin Reduction Potential in Bacterial Morphinone Reductase^*

Hanan L. Messiha, Neil C. Bruce, Benedict M. Sattelle, Michael J. Sutcliffe, Andrew W. Munro¶, and Nigel S. Scrutton||

From the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH and the Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom

The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.

Received for publication, March 1, 2005 , and in revised form, May 13, 2005.

^* This work was supported in part by the UK Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ A Royal Society Leverhulme Trust Senior Research Fellow.

^|| To whom correspondence should be addressed. Tel.: 44-116-229-7071; Fax: 44-166-252-3369; E-mail: nss4{at}le.ac.uk.

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