Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies
Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies
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DOI：
10.1093/oxfordjournals.jbchem.a021837
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发表时间：
1997-10-01
影响因子：
2.7
通讯作者：
Maeshima, M
中科院分区：
文献类型：
--
作者：
Takasu, A;Nakanishi, Y;Maeshima, M
液泡型H~+转运无机焦磷酸酶是一种以简单物质为能量供体的单一蛋白酶，利用亲水环和羧基末端对应的多肽(DVGADLVGKVE)的抗体研究了该酶的功能域，前者的抗体与不同植物的焦磷酸酶发生明显的反应，并强烈抑制纯化的酶的水解性和膜泡的质子泵活性。这些结果表明，该序列是一个实际的底物结合部位，是一个共同的基序，针对羧基末端的抗体只与绿豆酶反应。抑制其水解性和质子泵活性，结果表明，H+-焦磷酸酶的羧基末端暴露在胞浆中且靠近催化部位，H+-焦磷酸酶对三磷酸和四磷酸的低速水解率，植酸、六磷酸肌醇即使在有镁离子存在的情况下也对酶有抑制作用，其50%抑制浓度为0.15 mM，二环己基二亚胺对H+-PPase的抑制作用被镁离子部分逆转。
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.