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，并于几年前开始了丙烯酰胺的工业生产。与应用工作的高度发展相比，腈水合酶的基础研究进展并不充分。因此，目前的工作重点是更基础的工作，阐明腈水合酶的辅因子和反应机制。总结如下结果：(1)腈水合酶有两种，铁或钴作为辅助因子。(2)ESR研究表明：腈水合酶是第一个已知的非血红素铁酶，具有典型的低自旋Fe(III)配位环境，天然酶中铁(III)位点的轴向位置可能被硫醇基和equo基团占据，脂肪族腈底物直接与通过水底物置换铁 (III) 活性中心。钴也可能起到相同的作用，即腈的结合和活化。(3)铁腈水合酶涉及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之间可能存在相互作用，例如氢键。(4)克隆了铁和钴腈水合酶。确定了这些基因的序列，发现尽管协同因子存在差异，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.