Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.
Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
J
J
Weber
S
S
Wilke-Mounts
S T
ST
Hammond
A E
AE
Senior
D000227:Adenine Nucleotides / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D000227:Adenine Nucleotides; D008024:Ligands; D000266:Adenylyl Imidodiphosphate; D014364:Tryptophan; D006180:Proton-Translocating ATPases