Takasu A, Nakanishi Y, Yamauchi T, Maeshima M
Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University.
J Biochem. 1997 Oct;122(4):883-9. doi: 10.1093/oxfordjournals.jbchem.a021837.
Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15 mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.
液泡H⁺转运无机焦磷酸酶是一种单蛋白酶，以一种简单物质作为能量供体。通过使用针对亲水环和羧基末端部分中与假定底物结合位点（DVGADLVGKVE）相对应的肽段的特异性抗体，研究了该酶的功能结构域。针对前一个肽段的抗体与不同植物物种的焦磷酸酶明显发生反应，并强烈抑制纯化酶的水解活性和膜囊泡的质子泵活性。这些结果表明该序列起到实际底物结合位点的作用，并且是一个共同基序。针对羧基末端部分的抗体仅与绿豆酶发生反应，抑制其水解和质子泵活性。结果表明羧基末端暴露于细胞质溶胶中且靠近催化位点。H⁺焦磷酸酶以低速率水解三磷酸和四磷酸。肌醇六磷酸即使在存在Mg²⁺的情况下也能抑制该酶。50%抑制浓度为0.15 mM。二环己基二亚胺对H⁺焦磷酸酶的抑制作用部分被Mg²⁺逆转。文中讨论了该酶的催化位点和膜拓扑结构。
