Noncatalytic nucleotide sites of Escherichia coli F1-ATPase were studied by site-directed mutagenesis, covalent photolabeling with 2-azido-ATP, and lin-benzo-ATP binding. In wild-type, 89% of 2-azido-ATP label was bound to β-subunit, whereas in the βY354F mutant, 95% of the label was bound to α- subunit. In the αR365Y mutant, label was seen on both α (38%) and β (62%); whereas in the αR365F mutant, 93% was on β. The fluorescence of noncatalytic site-bound lin-benzo-ATP was quenched markedly in F1 from wild- type (76% quench), αR365F (85%), αR365Y (90%), and αR365Y,βY354F (83%), but only by 28% in βY354F. These results together demonstrate that residues αR365 and βY354 lie close to the base moiety of adenine nucleotide bound in F1 noncatalytic sites. From comparison of sequences of α- and β-subunits, it appears that residue αR365 in noncatalytic sites is equivalent to residue βY331 of the catalytic sites. Two unintended mutants were obtained in which α-subunit was increased in length by 17 amino acids due to repeat of residues α361 to α377, with either F or Y in the repeated α365 position. Soluble F1 was obtained from both mutants, with novel properties.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|