Azidonaphthoyl-ADP: a specific photolabel for the high-affinity nucleotide-binding sites of F1-ATPase.
3'-O-[5-azidonaphthoyl]-ADP has been synthesized as a photoreactive analog to 3'-O-naphthoyl(1)-ADP which is known to bind to the high-affinity nucleotide sites of mitochondrial F1-ATPase, considered to be the catalytic sites. The photolabel in the dark acts as a ligand to F1-ATPase and as a competitive inhibitor with Ki = 11 microM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy of 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both alpha and beta polypeptides of F1; it is accompanied by corresponding removal of reversible high-affinity sites for ADP or naphthoyl-ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D008929:Mitochondria, Heart / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods
D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; D000244:Adenosine Diphosphate; C043011:3'-O-(5-azidonaphthoyl)adenosine diphosphate; C043012:5-azidonaphthoic acid; D006180:Proton-Translocating ATPases