3′-O-(4-Benzoyl)benzoyladenosine 5′-Triphosphate Inhibits Activity of the Vacuolar (H+)-ATPase from Bovine Brain Clathrin-coated Vesicles by Modification of a Rapidly Exchangeable, Noncatalytic Nucleotide Binding Site on the B Subunit

This is the authors' abstract. We don't add key points for this paper.

It was previously observed that the B subunit of the tonoplast V-ATPase 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 further characterized the nucleotide binding sites on the V-ATPase and the interaction between BzATP and the B subunit. We observe that the V-ATPase isolated from bovine clathrin-coated vesicles possesses approximately 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 noncovalent inhibitor in the absence of irradiation, changing the kinetic characteristics of ATP hydrolysis. Irradiation of the V-ATPase in the presence of [3H]BzATP results primarily in modification of the 58-kDa B subunit, with complete inhibition of V-ATPase activity occurring upon modification of one B subunit per V-ATPase complex. Inhibition occurs as the result of modification of a rapidly (t1/2 < 2 min) exchangeable site, and yet this site does not correspond to a catalytic site, as indicated by the effects of cysteine-modifying reagents which react with Cys254 located at the catalytic sites on the A subunit. Thus, the noncatalytic nucleotide binding site modified by BzATP appears to be rapidly exchangeable. The site of [3H]BzATP modification of the B subunit was localized to the region Ile164 to Gln171, which from the x-ray crystal structure of the homologous F-ATPase alpha subunit, is within 10 A of the ribose ring of ATP bound to the noncatalytic nucleotide binding site. Thus, despite the absence of a glycine-rich loop region in the B subunit, these data are consistent with a similar overall folding pattern for the V-ATPase B subunit and the F-ATPase alpha subunit.