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.
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.
Vasilyeva
E
E
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Forgac
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000187:drug effects* / Q000201:enzymology; D002384:Catalysis; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009711:Nucleotides / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014316:Tritium; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology
D002966:Clathrin; D004791:Enzyme Inhibitors; D009711:Nucleotides; D014316:Tritium; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases