Unguryte A, Smirnova I N, Baykov A A
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Arch Biochem Biophys. 1989 Sep;273(2):292-300. doi: 10.1016/0003-9861(89)90487-6.
Initial rates of PPi hydrolysis by cytosolic and mitochondrial inorganic pyrophosphatases of rat liver have been measured in the presence of 0.2-100 microM MgPPi and 0.01-50 mM Mg2+ at pH 7.2 to 9.3. The apparently simplest model consistent with the data for both enzymes implies that they bind substrate, in the form of MgPPi, and three Mg2+ ions, of which two are absolutely required for activity. The third metal ion facilitates substrate binding but decreases maximal velocity for the cytosolic enzyme, while substrate binding is only modulated for the mitochondrial enzyme. The model is also applicable to bovine heart mitochondrial pyrophosphatases. The active form of the substrate for the cytosolic pyrophosphatase is MgP2O7(-2); the catalytic and metal-binding steps require a protonated group with pKa = 9.2 and an unprotonated group with pKa = 8.8, respectively. The results indicate that the mitochondrial pyrophosphatase is more sensitive to variations of Mg2+ concentration in rat liver cells than is the cytosolic one.
在pH 7.2至9.3的条件下，于0.2 - 100微摩尔/升的MgPPi和0.01 - 50毫摩尔/升的Mg²⁺存在时，测定了大鼠肝脏胞质和线粒体无机焦磷酸酶催化PPi水解的初始速率。与两种酶的数据相符的看似最简单的模型表明，它们以MgPPi的形式结合底物以及三个Mg²⁺离子，其中两个是活性绝对必需的。第三个金属离子促进底物结合，但降低了胞质酶的最大反应速度，而底物结合仅对线粒体酶有调节作用。该模型也适用于牛心线粒体焦磷酸酶。胞质焦磷酸酶的底物活性形式为MgP₂O₇⁻²；催化步骤和金属结合步骤分别需要一个pKa = 9.2的质子化基团和一个pKa = 8.8的未质子化基团。结果表明，大鼠肝细胞中的线粒体焦磷酸酶比胞质焦磷酸酶对Mg²⁺浓度变化更敏感。
