3'-O-(4-BENZOYL)BENZOYLADENOSINE 5'-TRIPHOSPHATE INHIBITS ACTIVITY OFTHE VACUOLAR (H-ATPASE FROM BOVINE BRAIN CLATHRIN-COATED VESICLES BY MODIFICATION OF A RAPIDLY EXCHANGEABLE, NONCATALYTIC NUCLEOTIDE-BINDING SITE ON THE B-SUBUNIT())

It was previously observed that the B subunit of the tonoplast V-ATPas e is modified by the photoactivated nucleotide analog 3'-O-(4-benzoyl) benzoyladenosine 5'-triphosphate (BzATP) (Manolson, M. F., Rea, P. A., and Poole, R. J. (1985) J. Biol. Chem. 260, 12273-12279). We have fur ther characterized the nucleotide binding sites on the V-ATPase and th e interaction between BzATP and the B subunit. We observe that the V-A TPase isolated from bovine clathrin coated vesicles possesses approxim ately 1 mol of endogenous, tightly bound ATP/mol of V-ATPase complex. BzATP is not a substrate for the V-ATPase, but does act as a noncovale nt inhibitor in the absence of irradiation changing the kinetic charac teristics of ATP hydrolysis. Irradiation of the V-ATPase in the presen ce of [H-3]BzATP results primarily in modification of the 58-kDa B sub unit, with complete inhibition of V-ATPase activity occurring upon mod ification of one B subunit per V-ATPase complex. Inhibition occurs as the result of modification of a rapidly (t(1/2) < 2 min) exchangeable site, and yet this site does not correspond to a catalytic site, as in dicated by the effects of cysteine-modifying reagents which react with Cys(254) located at the catalytic sites on the A subunit. Thus, the n oncatalytic nucleotide binding site modified by BzATP appears to be ra pidly exchangeable. The site of [H-3]BzATP modification of the B subun it was localized to the region Ile(164) to Gln(171), which from the x- ray crystal structure of the homologous F-ATPase alpha subunit, is wit hin 10 Angstrom of the ribose ring of ATP bound to the noncatalytic nu cleotide binding site. Thus, despite the absence of a glycine-rich loo p region in the B subunit, these data are consistent with a similar ov erall folding pattern for the V-ATPase B subunit and the F-ATPase alph a subunit.