Misra I, Charlier H A, Miziorko H M
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Biochim Biophys Acta. 1995 Mar 15;1247(2):253-9. doi: 10.1016/0167-4838(94)00223-4.
The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6. The observation of optimal activity at alkaline pH and the insensitivity of pK to divalent cation concentration suggest that the pK reflects ionization of an amino-acid side chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization. Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by this mechanism-based inhibitor, stoichiometric modification occurs. Amino-acid analysis indicates that cysteine is the principal target in C129S enzyme, demonstrating the presence of a second reactive cysteine within this enzyme. To test whether another cysteine functions in reaction chemistry, conserved cysteines were identified by sequence homology analysis. Five cysteine residues (C59, C69, C224, C232, C268), invariant in the nine sequences available for various eukaryotic HMG-CoA synthase isozymes, were individually replaced by alanine in a series of mutant enzymes. Kinetic analyses of the isolated mutant HMG-CoA synthases indicate that none of these is crucial to the chemistry that results in production of HMG-CoA. These results further distinguish the HMG-CoA synthase reaction from the related condensation of acyl-CoA substrates catalyzed by beta-ketothiolase.
禽类胞质HMG-CoA合酶活性的pH依赖性符合滴定曲线，其pK = 8.6。在碱性pH下观察到最佳活性以及pK对二价阳离子浓度不敏感，这表明pK反映的是氨基酸侧链（如半胱氨酰巯基）的电离，而非底物的烯醇化。当3-氯丙酰辅酶A与HMG-CoA合酶C129S反应时（C129S是一种缺乏通常被这种基于机制的抑制剂靶向的巯基的酶变体），会发生化学计量的修饰。氨基酸分析表明，半胱氨酸是C129S酶中的主要靶点，这证明该酶中存在第二个反应性半胱氨酸。为了测试另一个半胱氨酸是否在反应化学中起作用，通过序列同源性分析鉴定了保守的半胱氨酸。在一系列突变酶中，将9种真核HMG-CoA合酶同工酶可用的9个序列中不变的5个半胱氨酸残基（C59、C69、C224、C232、C268）分别替换为丙氨酸。对分离出的突变型HMG-CoA合酶的动力学分析表明，这些残基对产生HMG-CoA的化学反应都不是关键的。这些结果进一步区分了HMG-CoA合酶反应与由β-酮硫解酶催化的相关酰基辅酶A底物缩合反应。
