Combined application of site-directed mutagenesis, 2-azido-ATP labeling, and lin-benzo-ATP binding to study the noncatalytic sites of Escherichia coli F1-ATPase.
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 beta-subunit, whereas in the beta Y354F mutant, 95% of the label was bound to alpha-subunit. In the alpha R365Y mutant, label was seen on both alpha (38%) and beta (62%); whereas in the alpha R365F mutant, 93% was on beta. The fluorescence of noncatalytic site-bound lin-benzo-ATP was quenched markedly in F1 from wild-type (76% quench), alpha R365F (85%), alpha R365Y (90%), and alpha R365Y, beta Y354F (83%), but only by 28% in beta Y354F. These results together demonstrate that residues alpha R365 and beta Y354 lie close to the base moiety of adenine nucleotide bound in F1 noncatalytic sites. From comparison of sequences of alpha- and beta-subunits, it appears that residue alpha R365 in noncatalytic sites is equivalent to residue beta Y331 of the catalytic sites. Two unintended mutants were obtained in which alpha-subunit was increased in length by 17 amino acids due to repeat of residues alpha 361 to alpha 377, with either F or Y in the repeated alpha 365 position. Soluble F1 was obtained from both mutants, with novel properties.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Lee
R S
RS
Wilke-Mounts
S
S
Grell
E
E
Senior
A E
AE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; C018225:benzo-ATP; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases