Baykov A A, Shestakov A S, Kasho V N, Vener A V, Ivanov A H
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Eur J Biochem. 1990 Dec 27;194(3):879-87. doi: 10.1111/j.1432-1033.1990.tb19482.x.
Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.
通过对焦磷酸水解与合成、磷酸与水之间的氧交换、酶结合焦磷酸的形成以及Mg2+结合的测量所获得的综合证据，使我们能够推断出在Mg2+存在下大肠杆菌无机焦磷酸酶的催化总体方案。我们确定了Mg2+与各种酶种类结合的平衡常数以及催化反应四个步骤的正向和反向速率常数，这四个步骤分别是PPi的结合/释放、PPi的水解/合成以及两个Pi分子的相继结合/释放。与面包酵母焦磷酸酶不同，大肠杆菌酶在两个方向上的催化都通过单一途径进行，这需要Mg2+与四种类型的位点结合。其中三个位点在没有底物的情况下可以被占据，并且在酶 - 底物复合物中其中一个位点对Mg2+的亲和力增加了两个数量级。交换过程中18O标记的磷酸同位素异构体的分布表明，活性位点中焦磷酸的水解明显是可逆的。通过直接测量估计该过程的平衡常数为5.0。焦磷酸水解和合成的最大速度之比为69。合成速率几乎完全由焦磷酸从酶中释放的速率决定。在水解反应中，酶结合的焦磷酸水解和两个磷酸分子的相继释放以几乎相等的速率常数进行。
