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https://hdl.handle.net/1959.11/14086
Title: | Increased Positive Electrostatic Potential in p-Hydroxybenzoate Hydroxylase Accelerates Hydroxylation but Slows Turnover | Contributor(s): | Otiz-Maldonado, Mariliz (author); Cole, Lindsay J (author); Dumas, Sara M (author); Entsch, Barrie (author); Ballou, David P (author) | Publication Date: | 2004 | DOI: | 10.1021/bi030193d | Handle Link: | https://hdl.handle.net/1959.11/14086 | Abstract: | Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding ρ-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates ρ-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of ρ-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site - to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of ρ-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) 'Proc. Natl. Acad. Sci. U.S.A. 99', 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis. | Publication Type: | Journal Article | Grant Details: | ARC/A09906323 | Source of Publication: | Biochemistry, 43(6), p. 1569-1579 | Publisher: | American Chemical Society | Place of Publication: | United States of America | ISSN: | 1520-4995 0006-2960 |
Fields of Research (FoR) 2008: | 060107 Enzymes | Socio-Economic Objective (SEO) 2008: | 970106 Expanding Knowledge in the Biological Sciences | Peer Reviewed: | Yes | HERDC Category Description: | C1 Refereed Article in a Scholarly Journal |
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Appears in Collections: | Journal Article |
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