Baykov A A, Hyytia T, Volk S E, Kasho V N, Vener A V, Goldman A, Lahti R, Cooperman B S
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Biochemistry. 1996 Apr 16;35(15):4655-61. doi: 10.1021/bi952635u.
Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .
在pH值6.0 - 10.0范围内，测定了大肠杆菌无机焦磷酸酶（E-PPase）催化焦磷酸（PPi）水解的稳态速率，该速率是焦磷酸镁（底物）和游离Mg2+离子（激活剂）的函数。通过将水解数据进行计算机拟合，并结合对Mg2+与酶结合的直接测量，得出了一个能够定量解释我们实验结果的模型。该模型的主要特点如下：（a）E-PPase催化作用在每个活性位点结合三个、四个（可能还有五个）Mg2+离子时均可进行；（b）催化作用需要一个必需碱和一个必需酸，这些基团的pKa值受结合的Mg2+化学计量比调节；（c）当游离Mg2+浓度>0.2 mM时，四金属途径占主导。该模型直接解释了几个活性位点变体中观察到的必需碱pKa增加与更高Mg2+浓度的活性要求之间的明显联系。通过对酶结合的PPi形成以及PPi水解、PPi合成和Pi-H2O氧交换速率的联合分析，在pH 6.5 - 9.3下测定了PPi - Pi平衡总体催化的微观速率常数。随着pH值升高，E-PPase在底物饱和时的催化活性朝着PPi水解方向增加，朝着PPi合成和Pi-H2O氧交换方向降低。这些变化分别主要是由于第二个释放的Pi解离速率增加以及酶结合的Pi合成酶结合的PPi速率降低所致。
