The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium.
The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 microM. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 microM. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with 3H. Sequence analysis of the labeled peptides revealed that one contained residues 423-441 of the beta subunit. A gap in position 2 of the sequence indicates that beta Phe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342-358 of the beta subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains beta Tyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that beta Tyr345 is labeled in this peptide.
Grodsky
N B
NB
Department of Chemistry and Biochemistry, University of Delaware 19716-0001, USA.
Allison
W S
WS
D000225:Adenine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010777:Photochemistry; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011804:Quinolines / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*
C407687:4-amino-1-octylquinaldinium; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011804:Quinolines; D006180:Proton-Translocating ATPases; D000225:Adenine