Pickart C M, Rose I A
J Biol Chem. 1986 Aug 5;261(22):10210-7.
Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.
泛素（Ub）羧基末端水解酶（E）催化多种C末端Ub衍生物在Ub羧基末端的水解反应。我们发现，仅当存在Ub时，该酶会被毫摩尔浓度的硼氢化钠或羟胺灭活。基于水解酶的作用机制是通过酰基-Ub-E中间体进行亲核催化这一假设，我们对这些结果进行了解释。被硼氢化钠灭活的酶具有以下特性。它是E和Ub的化学计量复合物，含有来自硼氢化[3H]钠的氚。该复合物在5M尿素的中性pH条件下稳定，可通过分子筛柱根据大小进行分离，但在更强的变性条件下会释放出Ub大小的标记产物。在酸性条件下释放的“Ub”是Ub羧基末端醛，这是基于以下观察结果：它含有还原复合物中存在的氚，并且能够由化学计量的新鲜酶形成无活性的酶，并且失活伴随着E-Ub加合物的形成；它具有醛所预期的化学性质：在用硼氢化[3H]钠对释放的Ub进行二次还原并完全酸水解后，在乙醇胺中发现了氚计数（Ub的羧基末端残基是甘氨酸）。这些结果表明，酶和Ub在平衡反应中结合形成酯或硫酯加合物（在Ub羧基末端），并且该加合物被硼氢化物捕获，形成非常稳定的无活性E-Ub（硫代）半缩醛，它无法进行第二步还原，并且可以在弱酸中释放Ub-醛。在水解酶存在羟胺的情况下，在酶水解1200个Ub-异羟肟酸分子的过程中会发生一次失活。在10 - 50mM羟胺范围内，水解/失活比是恒定的，这表明羟胺和水与之反应的E-Ub形式不同，且不是快速平衡的。根据硼氢化研究推断，无活性的酶可能是由E-Ub（硫醇）酯生成的E-Ub混合酸酐形成的酰基异羟肟酸。已开发出一种直接放射性测定法，使用[3H]丁醇-4-胺的Ub-C末端酰胺作为底物来测定水解酶活性。
