Berndt M C, Bowles M R, King G J, Zerner B
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Biochim Biophys Acta. 1996 Dec 5;1298(2):159-66. doi: 10.1016/s0167-4838(96)00117-3.
Identical Kcat values (approximately 40 s-1) are obtained for the chicken liver carboxylesterase catalyzed hydrolysis of phenyl, p-nitrophenyl and o-nitrophenyl benzoates providing support for the involvement of an acyl-enzyme pathway, with the rate-limiting deacylation of a common benzoyl-enzyme intermediate. Chicken liver carboxylesterase catalyzed fragmentation of (E)-benzilmonoxime O-2,4-dinitrophenyl ether shows a pH dependence on a group active in the free base form with a pK'a approximately 5.0. The Ki-pH profile for benzil inhibition shows a dependence on a similar group with a pK'a = 5.4. The reactions between chicken liver carboxylesterase and the hydrated aldehyde, chloral hydrate, have shown this compound to be at once a substrate and potent inhibitor of the enzyme. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is first dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent aldehyde produces a hemiacetal adduct. The Ki value for chloral hydrate inhibition calculated from the kinetic analysis (90 nM) compares favourably with the value measured from the steady-state kinetics (87 nM).
鸡肝羧酸酯酶催化水解苯甲酸苯酯、对硝基苯酯和邻硝基苯酯时，得到相同的催化常数（Kcat）值（约40 s-1），这为酰基酶途径的参与提供了支持，其中常见的苯甲酰酶中间体的脱酰基反应是限速步骤。鸡肝羧酸酯酶催化（E）-苯偶姻单肟O-2,4-二硝基苯醚的裂解显示，pH值对以游离碱形式存在的活性基团有依赖性，其pK'a约为5.0。苯偶姻抑制的Ki-pH曲线显示对类似基团有依赖性，pK'a = 5.4。鸡肝羧酸酯酶与水合醛水合氯醛之间的反应表明，该化合物既是该酶的底物又是强效抑制剂。抑制动力学与一种机制一致，即结合的水合物首先在酶催化的限速步骤中脱水。活性位点丝氨酸对母体醛的亲核攻击产生半缩醛加合物。根据动力学分析计算出的水合氯醛抑制的Ki值（90 nM）与从稳态动力学测得的值（87 nM）相当。
