An essential arginine residue in vacuolar H+-ATPase purified from etiolated mung bean seedlings

Treatments of the tonoplast ATPase purified from mung bean seedlings (Vigna radiata L.) with guanidino modifiers, phenylglyoxal and 2,3-butanedione, c aused a marked loss of the ATP hydrolysis activity and proton translocation in a concentration-dependent manner. Kinetic analysis yielded first order rate constants, k(2), of 0.416 and 0.227 s(-1) and steady-state dissociatio n constants, K-i, of 19.3 and 24.2 mM for phenylglyoxal- and butanedione-in hibition of vacuolar H+-ATPase, respectively. The reaction order of phenylg lyoxal- and butanedione-inhibition was calculated to be 0.94 and 0.89, resp ectively, suggesting that at least one arginine residue of vacuolar H+-ATPa se was modified by both reagents. Lineweaver-Burk plots showed that the mod e of inhibition of vacuolar H+-ATPase by both modifiers is competitive. Mg- ATP, the physiological substrate of vacuolar H+-ATPase, but not its analogs , exerted preferentially partial protection against phenylglyoxal and butan edione, indicating that the arginine residue involved in the inhibition of enzymatic activity may be located at or near the active site and directly p articipate in the binding of the substrate.