論文 - 表　弘志

ESCHERICHIA-COLI ATP SYNTHASE (F-ATPASE) - CATALYTIC SITE AND REGULATION OF H+ TRANSLOCATION 査読

M FUTAI, A IWAMOTO, H OMOTE, Y ORITA, K SHIN, RK NAKAMOTO, M MAEDA

JOURNAL OF EXPERIMENTAL BIOLOGY   172   443 - 449   1992年11月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that betaLys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in betaLys-155 or betaThr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues betaSer-174, betaGlu-192 and betaVal-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.

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EFFECTS OF MUTATIONS OF CONSERVED LYS-155 AND THR-156 RESIDUES IN THE PHOSPHATE-BINDING GLYCINE-RICH SEQUENCE OF THE F1-ATPASE-BETA SUBUNIT OF ESCHERICHIA-COLI 査読

H OMOTE, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY   267 ( 29 )   20571 - 20576   1992年10月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

BetaLys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of betaLys- 155 and betaThr-156, mutants (betaLys-155 --> Ala, Ser, or Thr; betaThr-156 --> Ala, Cys, Asp, or Ser; betaLys-155/betaThr-156 --> betaThr-155/betaLys-156; and betaThr-156/betaVal-157 --> betaAla-156/betaThr-157) were constructed, and their properties were studied extensively. The betaSer-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had <0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (betaAla-155, betaSer-155, betaAla-156, and betaCys-156) showed low rates of multisite (<0.02% of the wild type) and unisite (<1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the betaAla-156 and betaCys-156 enzymes and 10(2)-fold lower with the betaAla-155 and betaSer-155 enzymes. The betaThr-156 --> Ala or Cys enzyme showed an altered response to Mg2+, suggesting that betaThr-156 may be closely related to Mg2+ binding. These results suggest that betaLys-155 and betaThr-156 are essential for catalysis and are possibly located in the catalytic site, although betaThr-156 could be replaced by a serine residue.

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ESCHERICHIA-COLI F0F1-ATPASE - RESIDUES INVOLVED IN CATALYSIS AND COUPLING 査読

RK NAKAMOTO, K SHIN, A IWAMOTO, H OMOTE, M MAEDA, M FUTAI

ION-MOTIVE ATPASES : STRUCTURE, FUNCTION, AND REGULATION   671   335 - 344   1992年

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：NEW YORK ACAD SCIENCES  

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MUTATIONS IN SER174 AND THE GLYCINE-RICH SEQUENCE (GLY149, GLY150, AND THR156) IN THE BETA-SUBUNIT OF ESCHERICHIA-COLI H+-ATPASE 査読

A IWAMOTO, H OMOTE, H HANADA, N TOMIOKA, A ITAI, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY   266 ( 25 )   16350 - 16355   1991年9月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A sequence motif in the beta-subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta-Gly149 by Ser suppressed the effect of the beta-Ser174 --> Phe mutation (defective H+-ATPase), but beta-Gly150 --> Ser substitution did not have this effect. A single mutation (beta-Gly149 --> Ser or beta-Gly150 --> Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta-Gly149 --> Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca2+-dependent activity, but had the wild-type level of Mg2+-dependent activity with active oxidative phosphorylation. Introduction of a beta-Gly149 --> Ser or beta-Gly150 --> Ser mutation with the beta-Ser174 --> Phe mutation also lowered the Ca2+-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta-Thr156 --> Ala or Cvs mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity.
The possible location of the catalytic site including divalent cation binding site(s) is discussed.

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