The ADP that binds tightly to nucleotide-depleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site.
Previous studies have shown that the initial complex formed when ADP binds to nucleotide-depleted F1-ATPase is transformed with a half time of 2 to 3 min to form with a much lower rate of ADP release. The ADP binding results in a strong inhibition of ATPase activity. The present paper reports appraisal of where the inhibitory ADP binds by use of the photoreactive ADP analog, 2-N3-ADP. In presence of Mg2+ the 2-N3-ADP like ADP induces reversible inhibition of nucleotide-depleted F1 (ndF1) with a Kd of about 10 nM. Photoirradiation of the inactive 2-N3-[beta-32P]ADP-ndF1 complex results in labeling of only the beta-subunit. The major labeled peptide isolated from a trypic digest consists of residues from Ala-338 to Arg-356, with Tyr-345 as the site of labeling. This identifies the site of the inhibitory ADP binding as one of the catalytic sites of the enzyme.
Milgrom
Y M
YM
Department of Chemistry and Biochemistry, University of California, Los Angeles.
Boyer
P D
PD
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D009249:NADP; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases