Zhou J, Adams J A
Department of Chemistry, San Diego State University, California 92182-1030, USA.
Biochemistry. 1997 Mar 11;36(10):2977-84. doi: 10.1021/bi9619132.
The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.
在丝氨酸特异性蛋白激酶（cAMP依赖性蛋白激酶）活性位点的天冬氨酸羧基，在X射线晶体结构中靠近肽底物的羟基质子（马杜苏丹等人，1994年），这表明该残基可能在磷酰基转移反应中作为通用碱催化剂。事实上，在这方面已经提出了几种建议。我们使用快速淬灭流动技术测量了该酶的预稳态动力学，以了解这种假定碱的作用。cAMP依赖性蛋白激酶对肽底物GRTGRRNSI的磷酸化表现出“爆发”动力学，这与肽快速磷酸化（154 s⁻¹）而产物缓慢释放（16 s⁻¹）的机制一致。用Mn²⁺替代Mg²⁺导致观察到的“爆发”速率常数降低13倍，这表明该瞬态要么受磷酰基转移步骤限制，要么受金属离子依赖性构象变化步骤限制。在两种二价金属离子存在下监测了重水对预稳态动力学的影响，在“爆发”阶段均未测量到溶剂同位素效应。在任何一种金属离子存在下，对kcat均观察到较大的溶剂同位素效应，并且在Mg²⁺存在下的质子累积分析表明在产物释放步骤中有两个或更多质子转移。最后，在6 - 9的pH范围内，使用Mg²⁺或Mn²⁺时，未观察到“爆发”速率常数对pH的依赖性。如果“爆发”阶段完全受磷酰基转移步骤限制，综合数据不支持涉及pKa大于5或小于10的通用碱催化剂的机制。如果“爆发”阶段受金属离子依赖性构象变化步骤限制，则磷酰基转移步骤的测量会被掩盖，并且碱催化剂的参与情况不确定。
