Baykov A A, Bakuleva N P, Rea P A
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Eur J Biochem. 1993 Oct 15;217(2):755-62. doi: 10.1111/j.1432-1033.1993.tb18303.x.
The results of analyses of the steady-state kinetics of the vacuolar H(+)-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata (mung bean) and purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) and K+ concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg2+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady-state kinetics of PPi hydrolysis. Two distinct Mg(2+)-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cations and Mg2+ and K+ do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pKa 5.7 and 8.6 in substrate binding and groups of pKa 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.
对从绿豆黄化下胚轴分离的原生质体膜泡的液泡H⁺转运焦磷酸酶（V-PPase）以及来自同一来源的纯化酶在广泛的Mg²⁺、焦磷酸（PPi）和K⁺浓度下的稳态动力学分析结果，与一个最小反应方案一致，其中焦磷酸二镁是活性底物种类，催化作用由预先形成的酶-Mg²⁺复合物介导。考虑到V-PPase对离子强度的敏感性，不需要额外的动力学相互作用来描述该酶的行为。N-乙基马来酰亚胺保护试验表明，在没有PPi的情况下Mg²⁺结合的解离常数比从PPi水解的稳态动力学估计的值小一个数量级。因此提出了两个不同的Mg²⁺结合位点。由于Mg²⁺的保护作用与单价阳离子的性质无关，并且Mg²⁺和K⁺在底物水解过程中不竞争，所以得出二价和单价阳离子在不同位点结合的结论。动力学参数的pH依赖性与pKa为5.7和8.6的基团参与底物结合以及pKa为6.1和9.0的基团参与底物转化步骤一致，表明至少四个可电离基团对催化至关重要。就V-PPase的反应机制以及体内胞质游离Mg²⁺和K⁺的潜在调节意义对这些发现进行了讨论。
