Le. Hunt et D. Sanders, THE KINETICS OF N-ETHYLMALEIMIDE INHIBITION OF A VACUOLAR H-ATPASE AND DETERMINATION OF NUCLEOTIDE DISSOCIATION-CONSTANTS(), Plant physiology, 110(1), 1996, pp. 97-103
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.