Studies on the cofactors and reaction mechanisms of nitrile hydratase which is useful for the synthesis of amides

我们已经建立了利用微生物腈水合酶酶法生产丙烯酰胺的工艺，并于几年前开始工业化生产丙烯酰胺。与腈水合酶应用研究的迅速发展相比，腈水合酶的基础研究进展并不充分。因此，目前的工作集中在更基础的工作，辅因子和腈水合酶的反应机理的阐明。总结了以下结果。(1)有两种腈水合酶，铁或钴作为辅因子参与。(2)ESR研究表明：腈水合酶是第一个已知的具有典型的低自旋Fe（III）配位环境的非血红素铁酶，在天然酶中的铁（III）位点的轴向位置可能被硫醇盐和equo基团占据，脂肪族营养底物通过水底物置换直接结合到铁（III）活性中心。钴也可能发挥同样的作用，腈的结合和活化。(3)铁腈水合酶涉及一个活性羰基辅因子作为第二辅基，可能是吡咯喹啉醌。在铁位点和水合PQQ的形式之间可能存在相互作用，例如氢键。(4)克隆了铁腈水合酶和钴腈水合酶。对这两个基因的序列进行了测定，发现尽管同源因子不同，但两个基因具有极高的同源性。

We have established, the enzymatic production process of acrylamide using the microbial nitrile hydratase and the industrial production of acrylamide was started since several years ago. Compared to the high development of the applied works, the basic studies on nitrile hydratase do not progress fully. Thus, the present works focused on more basic works, the elucidation of the cofactors and reaction mechanisms of nitrile hydratase. The following results are summarized.(1) There are two kinds of nitrile hydratases, iron or cobalt is involved as a cofactor.(2) ESR studies showed the following : nitrile hydratase is the first known nonheme iron enzyme with a typical low-spin Fe (III) coordination environment, the axial position of the iron (III) site in the native enzyme may be occupied by thiolate and equo groups, and aliphatic nutrile-substrates directly bind to the iron (III)-active center through water substrate replacement. Cobalt also probably plays the same role, the binding of nitrile and activation of it.(3) Iron nitrile hydratase involves an active carbonyl cofactor as a second prosthetic group, probably pyrrologuinoline quinone. There may be an interaction, such as a hydrogen bond, between the ferric site and a form of hydrated PQQ.(4) The iron and cobalt nitrile hydratases were cloned. The sequence of these genes was determined and the extreme high homology of two genes was found in spite of the difference of confactors.