Hiraishi H, Yokoi F, Kumon A
Department of Biochemistry, Saga Medical School, Japan.
Arch Biochem Biophys. 1998 Jan 15;349(2):381-7. doi: 10.1006/abbi.1997.0480.
A 56-kDa inorganic pyrophosphatase isolated from bovine liver hydrolyzed PPi, imidodiphosphate, 3-phosphohistidine, and 6-phospholysine at rates of 0.11, 0.44, 1.09, and 1.22 mumol/min/mg protein, respectively, in a reaction mixture containing 1 mM MgCl2 at pH 8.2. The hydrolysis of imidodiphosphate was influenced by various treatments in a different manner from that of N-phosphorylated amino acids, indicating that the pyrophosphatase has two different catalytic sites for imidodiphosphate and N-phosphorylated amino acids, respectively. Evidence for separate catalytic sites consists of the following findings: the activity on hydrolysis of imidodiphosphate gave a bell-shaped pH curve with a peak at pH 6.5, while the activity on hydrolysis of N-phosphorylated amino acids maintained a high level in the pH range between 6.0 and 9.5. One hundred micromolar p-chloromercuriphenyl sulfonate inhibited the hydrolysis of imidodiphosphate by 35% and did not inhibit that of N-phosphorylated amino acids. Two millimolar magnesium chloride repressed the hydrolysis of imidodiphosphate and had no inhibitory effect on the hydrolysis of N-phosphorylated amino acids. Moreover, methylenediphosphonic acid, an analog of imidodiphosphate, stimulated the hydrolysis of imidodiphosphate in the presence of MgCl2, while it potentiated the substrate inhibition on hydrolysis of N-phosphorylated amino acids.
从牛肝脏中分离出的一种56千道尔顿的无机焦磷酸酶，在pH 8.2、含有1 mM氯化镁的反应混合物中，分别以0.11、0.44、1.09和1.22 μmol/分钟/毫克蛋白质的速率水解焦磷酸、亚氨基二磷酸、3-磷酸组氨酸和6-磷酸赖氨酸。亚氨基二磷酸的水解受到各种处理的影响，其方式与N-磷酸化氨基酸不同，这表明该焦磷酸酶分别具有针对亚氨基二磷酸和N-磷酸化氨基酸的两个不同催化位点。存在单独催化位点的证据包括以下发现：亚氨基二磷酸水解的活性给出了一条钟形pH曲线，在pH 6.5处有一个峰值，而N-磷酸化氨基酸水解的活性在6.0至9.5的pH范围内保持较高水平。100 μM对氯汞苯磺酸盐抑制亚氨基二磷酸的水解35%，但不抑制N-磷酸化氨基酸的水解。2 mM氯化镁抑制亚氨基二磷酸的水解，对N-磷酸化氨基酸的水解没有抑制作用。此外，亚氨基二磷酸的类似物亚甲基二膦酸在氯化镁存在下刺激亚氨基二磷酸的水解，而它增强了对N-磷酸化氨基酸水解的底物抑制作用。
