Entsch B, van Berkel W J
Department of Biochemistry and Microbiology, University of New England, Armidale, N.S.W., Australia.
FASEB J. 1995 Apr;9(7):476-83. doi: 10.1096/fasebj.9.7.7737455.
Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein involved in degradation of aromatic compounds, and it has become a model for enzymes involved in the oxygenation of a substrate. The chemical and kinetic mechanisms of this enzyme are described and integrated with an outline of the structure of the protein from crystallographic analysis. The structure is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction. Recently, mechanistic studies of site-directed mutants, combined with structural analyses, have provided some exciting discoveries about protein function. The substrate during catalysis is largely isolated from solvent in the active site, a necessary condition for successful product formation. The flavin ring structure moves substantially in the active site, probably to enable substrate and product exchange into this site and possibly to regulate the reduction of the flavin by NADPH. A chain of H-bonds can connect p-hydroxy-benzoate in the active site of the enzyme with the protein surface. This chain is responsible for the reversible formation of substrate phenolate anion observed in the active site and partly responsible for the reactivity of this substrate.
对羟基苯甲酸羟化酶（EC 1.14.13.2）是一种黄素蛋白，参与芳香族化合物的降解，并且已成为涉及底物氧化的酶的模型。描述了该酶的化学和动力学机制，并与晶体学分析得出的蛋白质结构概述相结合。该结构不同寻常，因为在反应中没有可识别的用于结合NADPH的结构域。最近，定点突变体的机制研究与结构分析相结合，为蛋白质功能提供了一些令人兴奋的发现。催化过程中的底物在很大程度上与活性位点中的溶剂隔离，这是成功形成产物的必要条件。黄素环结构在活性位点中大幅移动，可能是为了使底物和产物能够进入该位点并可能调节NADPH对黄素的还原。一条氢键链可将酶活性位点中的对羟基苯甲酸与蛋白质表面相连。这条链负责在活性位点中观察到的底物酚盐阴离子的可逆形成，并部分负责该底物的反应性。
