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-protei n enzyme and uses a simple substance as an energy donor, Functional do mains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLV GKVE) in the hydrophilic loop and the carboxyl terminal part, The anti body to the former peptide clearly reacted with the pyrophosphatases o f different plant species, and strongly inhibited the hydrolytic activ ity of the purified enzymes and the proton pumping activity of membran e vesicles, These results indicate that the sequence functions as an a ctual substrate-binding site and is a common motif, The antibody to th e carboxyl terminal part reacted only to the mung bean enzyme, suppres sing 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 tet raphosphate at low rates, Phytic acid, myo-inositol hexaphosphate, inh ibited the enzyme even in the presence of Mg2+, The concentration for 50% inhibition was 0.15 mM, The inhibition of H+-PPase by dicyclohexyl diimide was partly reversed by Mg2+, The catalytic site and the membra ne topology of the enzyme are discussed.