title: Domains near ATP gamma phosphate in the catalytic site of H+-ATPase. Model proposed from mutagenesis and inhibitor studies.
abstract: The beta Gly-149 residue is in a glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr; residues 149-156) of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit. Substitution of beta Gly-149 by Ser suppressed the effect of the beta Ser-174-->Phe mutation (Iwamoto, A., Omote, H., Hanada, H., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1991) J. Biol. Chem. 266, 16350-16355), suggesting that beta Gly-149 is located near beta Ser-174. In this study, we introduced different residues at position 149 and found that a single mutant beta Cys-149 was defective. The effect of beta Cys-149 mutation was suppressed by beta Gly-172-->Glu, beta Ser-174-->Phe, beta Glu-192-->Val, or beta Val-198-->Ala replacement. These results suggest that beta Gly-149, beta Gly-172, beta Ser-174, beta Glu-192, and beta Val-198 residues are located close together in the catalytic site. From these findings we propose a model of the catalytic site of the enzyme near the gamma phosphate moiety of ATP. F1 enzymes with the double mutations beta Cys-149/beta Glu-172, beta Cys-149/beta Phe-174, beta Cys-149/beta Val-192, and beta Cys-149/beta Ala-198 were less sensitive than wild-type F1 to dicyclohexylcarbodiimide and adenosine triphosphopyridoxal (an affinity analogue of ATP forming a Schiff base with the epsilon-amino group of beta Lys-155 or beta Lys-201), and became sensitive to N-ethylmaleimide in an ATP-protected manner. These results of inhibitor studies are consistent with the proposed model.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Iwamoto
authors.forename: M Y
authors.initials: MY
authors.lastname: Park
authors.forename: M
authors.initials: M
authors.lastname: Maeda
authors.forename: M
authors.initials: M
authors.lastname: Futai
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C049517:adenosine triphosphopyridoxal; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide