Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies.
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.
Takasu
A
A
Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University.
Nakanishi
Y
Y
Yamauchi
T
T
Maeshima
M
M
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002384:Catalysis; D007887:Fabaceae / Q000201:enzymology*; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D017136:Ion Transport; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011522:Protons; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology*
D000906:Antibodies; D011522:Protons; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase