Kobayashi M, Goda M, Shimizu S
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Biochem Biophys Res Commun. 1998 Dec 30;253(3):662-6. doi: 10.1006/bbrc.1998.9834.
While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically. This nitrilase exhibited a Km of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the Vmax for benzamide was six orders of magnitude lower than that for benzonitrile. Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner. A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity. This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.
虽然据报道酰胺作为迄今为止所报道的所有腈水解酶的底物完全无活性，但来自红平红球菌J1的腈水解酶，它催化腈中C-N三键的水解裂解以形成酸和铵，令人惊讶地发现它能以化学计量的方式催化酰胺水解为酸和铵。这种腈水解酶对苯甲酰胺的Km为2.94 mM，与作为原始底物的苄腈的Km（2.10 mM）相似，但苯甲酰胺的Vmax比苄腈的Vmax低六个数量级。苯甲酰胺以可逆的、明显竞争性的方式抑制腈水解酶反应。一种含有丙氨酸或丝氨酸而非对腈水解酶催化活性至关重要的Cys165的突变腈水解酶没有酰胺酶活性。这一观察结果表明Cys165在酰胺以及腈的水解中起关键作用。连同一些先前指出某些腈水解酶会从腈中产生少量酰胺作为副产物的报道，上述意外发现表明在以腈或酰胺为底物的腈水解酶反应中存在一个共同的四面体中间体。
