THE KINETICS OF N-ETHYLMALEIMIDE INHIBITION OF A VACUOLAR H-ATPASE AND DETERMINATION OF NUCLEOTIDE DISSOCIATION-CONSTANTS()

All eukaryotic vacuolar (V-type) ATPases share the property of being i nhibited by low concentrations (1-2 mu M) of N-ethylmaleimide (NEM). T his distinguishes them from P-type ATPases, which are inhibited by hig her concentrations of NEM (0.1-1 mM), and F-type ATPases, which are vi rtually resistant to inhibition by NEM. Using tonoplast vesicles from Beta vulgaris we have determined the kinetics of NEM inactivation of t he V-type ATPase to be pseudo-first order. The concentration dependenc e of the reaction indicates interaction with a single class of inhibit ory site with a rate constant of 4.1 x 10(4) M(-1) min(-1). Nucleotide s protect against inactivation with an efficacy that agrees with their capacity to act as enzyme substrates. The dissociation constant for M gATP has been determined from protection experiments to be 0.44 mM, wh ich is close to the observed K-m for hydrolysis (0.39 mM). Likewise, t he dissociation constant for protection by MgADP (127 mu M) is close t o its inhibition constant as a competitive inhibitor (110 mu M). Taken together, these findings suggest that NEM inactivation is associated with nucleotide protectable exposure of a single cysteine residue on t he catalytic subunit and confirm the utility of this residue for the d etermination of ligand dissociation constants through protection of ma leimide inhibition.