STRUCTURE-FUNCTION STUDIES OF SUBSTRATE OXIDATION BY BOVINE SERUM AMINE OXIDASE - RELATIONSHIP TO COFACTOR STRUCTURE AND MECHANISM
STRUCTURE-FUNCTION STUDIES OF SUBSTRATE OXIDATION BY BOVINE SERUM AMINE OXIDASE - RELATIONSHIP TO COFACTOR STRUCTURE AND MECHANISM
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DOI：
10.1021/bi00232a035
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发表时间：
1991-05-07
期刊：
影响因子：
2.9
通讯作者：
KLINMAN, JP
中科院分区：
文献类型：
--
作者：
HARTMANN, C;KLINMAN, JP
底物氧化的化学机制，催化牛血清胺氧化酶，已被探索的结构-反应性的相关性的详细调查。 过去的机制研究，涉及底物还原捕获到辅因子[Hartmann，C.，& Klinman，J. P.（1987）J.Biol.Chem.262，962]，暗示了底物亚胺络合物在催化氧化还原机理中的中间性。 这些研究提出了底物氧化的转氨机制，类似于磷酸吡哆醛依赖性酶。 在磷酸吡哆醛催化的反应中，转氨过程涉及共振稳定的碳负离子中间体的瞬时形成。 尽管已经提出了描述活性位点碱基参与牛血清胺氧化酶催化的证据[Farnum，M. F.、帕尔契奇湾M.，& Klinman，J. P.（1986）Biochemistry 25，1898]，衍生自C-H键裂解的中间体的性质尚未直接解决。 为了研究这个问题，使用一系列对位取代的苄胺进行结构反应性研究。 具有酶促反应的固有同位素效应的先验知识允许从稳态数据计算微观速率常数[Palcic，M. M.，和Klinman，J.P.（1983）Biochemistry 22，5957]。 确定所有底物的氘同位素对k（cat）和k（cat）/K（m）参数的影响，从而计算C-H键裂解的速率常数（k3）和底物解离常数（K（d））。 对乙酰基苄胺，p-（三氟甲基）苄胺，和未取代的苄胺获得的前稳态常数表现出良好的协议与稳态同位素效应计算值。 多元回归分析得到的电子效应rho = 1.47 +/- 0.27的键裂解步骤，支持的碳负离子物种的中间。 由回归分析确定的另外的效应表明疏水取代基对催化的抑制（pi = -0.71 +/-0.21）。 这些结果导致通过牛血清胺氧化酶中共价结合的辅因子6-羟基多巴氧化胺的反应机理[Janes，S. M.，Mu，D.，Wemmer，D.，史密斯，A. J.，考尔，S.，Maltby，D.，Burlingame，A. L.，和Klinman，J.P.（1990）Science 248，981]。
The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on k(cat) and k(cat)/K(m) parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (K(d)). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects. Multiple regression analysis yielded an electronic effect of rho = 1.47 +/- 0.27 for the bond cleavage step, supporting the intermediacy of a carbanion species. An additional effect, determined from regression analysis, indicated inhibition of catalysis by hydrophobic substituents (pi = -0.71 +/- 0.21). These results lead to a reaction mechanism for amine oxidation by the covalently bound cofactor in bovine serum amine oxidase, 6-hydroxydopa [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., & Klinman, J. P. (1990) Science 248, 981].