Woods M J, Findlater J D, Orsi B A
Biochim Biophys Acta. 1979 Mar 16;567(1):225-37. doi: 10.1016/0005-2744(79)90189-x.
The kinetic constants for hydrolysis and transfer (with hydroxylamine as the alternate acceptor) of the aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were determined for a variety of acetyl and propionyl derivatives. The results obtained were consistent with a ping-pong or substitution mechanism. Product inhibition, which was pH dependent, implicated an acyl-enzyme compound as a compulsory intermediate and indicated that ammonia combined additionally with the free enzyme in a dead-end manner. The uncompetitive activation of acetamide hydrolysis by hydroxylamine and the observation that the partitioning of products between acetic acid and acetohydroxamate was linearly dependent on the hydroxylamine concentration substantiated these conclusions and indicated that deacylation was at least partially rate limiting. With propionamide as the acyl donor apparently anomalous results, which included inequalities in certain kinetic constants and a hyperbolic dependence of the partition ratio on the hydroxylamine concentration, could be explained by postulating a compulsory isomerisation of the acyl-enzyme intermediate prior to the transfer reaction.
测定了铜绿假单胞菌脂肪族酰胺酶（酰基酰胺酰胺水解酶，EC 3.5.1.4）对多种乙酰基和丙酰基衍生物的水解及转移（以羟胺作为替代受体）的动力学常数。所得结果与乒乓机制或取代机制一致。产物抑制作用依赖于pH，这表明酰基酶化合物是一个必需中间体，并且表明氨以终产物的方式额外地与游离酶结合。羟胺对乙酰胺水解的反竞争性激活作用以及乙酸和乙酰氧肟酸之间产物分配与羟胺浓度呈线性相关的观察结果证实了这些结论，并表明脱酰化至少部分是限速步骤。以丙酰胺作为酰基供体时，出现了明显异常的结果，包括某些动力学常数的不相等以及分配比与羟胺浓度呈双曲线关系，这可以通过假定在转移反应之前酰基酶中间体存在必需的异构化来解释。
