title: Catalytic site of F1-ATPase of Escherichia coli. Lys-155 and Lys-201 of the beta subunit are located near the gamma-phosphate group of ATP in the presence of Mg2+.
abstract: The catalytic site of Escherichia coli F1 was probed using a reactive ATP analogue, adenosine triphosphopyridoxal (AP3-PL). For complete loss of enzyme activity, about 1 mol of AP3-PL bound to 1 mol of F1 was estimated to be required in the presence or absence of Mg2+. About 70% of the label was bound to the alpha subunit and the rest to the beta subunit in the absence of Mg2+, and the alpha Lys-201 and beta Lys-155 residues, respectively, were the major target residues (Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T. (1988) FEBS Lett. 233, 347-351). Addition of Mg2+ decreased the AP3-PL concentration required for half-maximal inhibition, and predominant labeling of the beta subunit (beta Lys-155 and beta Lys-201) with the reagent. ATP and ADP were protective ligands in the presence and absence of Mg2+. The alpha subunit mutation (alpha Lys-201----Gln or alpha Lys-201 deletion) were active in oxidative phosphorylation. However, purified mutant F1s showed impaired low multi-site activity, although their uni-site catalyses were essentially normal. Thus alpha Lys-201 is not a catalytic residue, but may be important for catalytic cooperativity. Mutant F1s were inhibited less by AP3-PL in the absence of Mg2+, and consistent with this, modifications of their alpha subunits by AP3-PL were reduced. AP3-PL was more inhibitory to the mutant enzymes in the presence of Mg2+, and bound to the beta Lys-155 and beta Lys-201 residues of mutant F1 (alpha Lys-201----Gln). These results strongly suggest that alpha Lys-201, beta Lys-155, and beta Lys-201 are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg2+.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ida
authors.forename: T
authors.initials: T
authors.lastname: Noumi
authors.forename: M
authors.initials: M
authors.lastname: Maeda
authors.forename: T
authors.initials: T
authors.lastname: Fukui
authors.forename: M
authors.initials: M
authors.lastname: Futai
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000235:genetics*; D008274:Magnesium; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D006180:Proton-Translocating ATPases; D008274:Magnesium; D008239:Lysine