Ortiz-Maldonado Mariliz, Cole Lindsay J, Dumas Sara M, Entsch Barrie, Ballou David P
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2004 Feb 17;43(6):1569-79. doi: 10.1021/bi030193d.
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 p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-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 p-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 p-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.
对羟基苯甲酸羟化酶是一种黄素蛋白单加氧酶，它催化的反应分为两个部分：响应对羟基苯甲酸与酶的结合，由NADPH还原酶辅因子FAD；还原型FAD与氧气氧化形成氢过氧化物，然后该氢过氧化物将对羟基苯甲酸氧化。这些不同的反应通过蛋白质结构中异咯嗪环的构象重排来协调。在本文中，我们通过酶突变Glu49Gln研究了活性位点正静电势增加对催化过程的影响。该突变从一个保守的埋藏电荷对中去除了一个负电荷。Glu49Gln突变酶的性质与活性位点正电势增加一致，但该突变酶难以研究，因为它不稳定。与野生型相比，突变酶的催化功能有两个重要变化。首先，瞬态形成的黄素氢过氧化物催化对羟基苯甲酸羟化的速率比野生型快一个数量级。这一结果与活性位点正电势的一个功能假设一致，即过氧化物键异裂时稳定负的C-4a-黄素醇盐离去基团。然而，突变酶比野生型酶是一种较差的催化剂，因为（与野生型不同）对羟基苯甲酸的结合是一个限速过程。我们的分析表明，突变酶在结合和释放底物所需的构象之间相互转化缓慢。我们得出结论，在Arg220Gln突变酶晶体中发现的新开放结构[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]对于催化过程中底物从氧化酶的结合和释放过程至关重要。
