Mechanistic studies of ubiquitin C-terminal hydrolase L1.
Mechanistic studies of ubiquitin C-terminal hydrolase L1.
复制标题
DOI：
10.1021/bi052135t
复制
发表时间：
2006-02-21
期刊：
影响因子：
2.9
通讯作者：
Stein, RL
中科院分区：
文献类型：
--
作者：
Case, A;Stein, RL
泛素C-末端水解酶（UCH）通过硫酯中间体切割Ub-X键（Ub是泛素，X是醇、胺或蛋白质），所述硫酯中间体由Cys-SH/His-Im催化二联体的Cys残基的亲核攻击产生。我们正在研究UCH-L1的机制，这是一种与帕金森病有关的UCH，现在希望报告我们的初步发现。(i)对UCH-L1催化的Ub-AMC（AMC，7-amido-4-methylcoumarin）水解反应的稳态前动力学研究表明，k（cat）受酰基酶形成的速率限制。因此，K-m = K-s，米氏络合物的解离常数，和k（cat）= k（2），酰基酶形成的速率常数。(ii)对于K-天冬氨酸（=K-s（-1）），Δ C-p = -0.8 kcal mol（-1）deg（-1），与底物缔合和酶构象变化之间的偶联一致。对于k（2），Δ S-双匕首= 0，表明在E-S中，底物和活性位点残基精确对齐以进行反应。(iii)溶剂同位素效应为D κ = 0.5和（D）k（2）= 0.9，表明底物结合于游离酶的形式，其中活性位点Cys以巯基形式存在。在所得米氏络合物中，二联体已互变异构为离子对Cys-S-/His-ImH（+）。硫醇盐的后续攻击产生酰基酶种类。与此相反，同位素效应的关联UCH-L1与过渡态类似物泛素醛表明，一个替代的机制途径，有时可以提供给UCH-L1涉及一般碱催化攻击Cys-SH的His-Im。
Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are Studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-finited by acyl-enzyme formation. Thus, K-m = K-s, the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K-assoc (=K-s(-1)), Delta C-p = -0.8 kcal mol(-1) deg(-1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), Delta S-double dagger = 0 and suggests that in the E-S, Substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are DKassoc = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S-/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.