Elucidation of the reaction mechanism of a PCB-degrading enzyme BphyC based on three-dimensional structural information.

所谓的BphC酶是一种双加氧酶，它可以裂解位于联苯及其衍生物中的儿茶酚环部分。裂解点位于邻苯二酚部分中相邻的两个羟基附近，因此酶以外二醇的方式起作用。这种酶利用分子氧进行这种反应，并在铁（2+）或铁离子的催化作用下结合原子氧原子。本文采用x射线结构分析方法对假单胞菌KKS102菌株BphC酶的三维结构进行了解析。生物学报。255,735-752(1996))。该酶的结构信息与野生和突变形式的各种酶动力学数据相结合，揭示了以下几点。关于催化机理：1)分子氧结合位点相对于结合底物分子的位置和取向是固定的，这似乎具体决定了环裂解的位置。2)其中一个保守残基His 194起催化碱从敏感羟基中提取氢的作用。底物特异性：1)底物结合位点本质上是疏水的，与底物的儿茶酚环部分具有很高的互补性。2)位于活性位点的铁离子对底物结合不一定是必需的（当然，对催化是必需的）。

The so-called BphC enzyme is a dioxygenase which cleaves the catechol ring moiety situated in biphenyl and its derivatives. The cleaving point is located adjacent to the two neighboring hydroxyl groups in a catechol moiety, thus the enzyme works in an extradiol fashion. This kind of enzyme performs such reactions making use of molecular oxygen and incorporates both the atomic oxygen atoms under the catalytic influence of Fe (2+) or ferrous ion.The present investigators solved the three-dimensional structure of a BphC enzyme from Pseudomonas sp. stran KKS102 by X-ray structure analysis (J.Mol. Biol. 255,735-752 (1996)). The structural information coupled with various enzyme kinetic data on wild and mutant forms of the enzyme revealed the following points.As for the catalytic mechanism :1) The site of ring cleavage appears to be specifically determined by the fact that the molecular oxygen binding site is fixed relative to the poisition and orientation of the bound substrate molecules.2) One of the conserved residues, His 194, plays a role of ctalytic base abstracting a hydrogen from the susceptible hydroxyl group.As for the substrate specificity :1) The substrate binding site is essentially hydrophobic exhibiting very high complimentarity to the catechool ring moiety of the substrates.2) The Fe ion situated in the active site is not necessarily essential for substrate binding (but essential for catalysis, of course).