TY - JOUR
T1 - Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase β subunit of Escherichia coli
AU - Omote, H.
AU - Maeda, M.
AU - Futai, M.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1992
Y1 - 1992
N2 - βLys-155 in the glycine-rich sequence of the β subunit of Escherichia coli F1-ATPase has been shown to be near the γ-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 βLys- 155 and βThr-156, mutants (βLys-155 → Ala, Ser, or Thr; βThr-156 → Ala, Cys, Asp, or Ser; βLys-155/βThr-156 → βThr-155/βLys-156; and βThr- 156/βVal-157 → βAla-156/βThr-157) were constructed, and their properties were studied extensively. The βSer-156 mutant was active in ATP synthesis and had ~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 (βAla- 155, βSer-155, βAla-156, and βCys-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 βAla-156 and βCys-156 enzymes and 102-fold lower with the βAla-155 and βSer-155 enzymes. The βThr-156 → Ala or Cys enzyme showed an altered response to Mg2+, suggesting that βThr-156 may be closely related to Mg2+ binding. These results suggest that βLys-155 and βThr-156 are essential for catalysis and are possibly located in the catalytic site, although βThr-156 could be replaced by a serine residue.
AB - βLys-155 in the glycine-rich sequence of the β subunit of Escherichia coli F1-ATPase has been shown to be near the γ-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 βLys- 155 and βThr-156, mutants (βLys-155 → Ala, Ser, or Thr; βThr-156 → Ala, Cys, Asp, or Ser; βLys-155/βThr-156 → βThr-155/βLys-156; and βThr- 156/βVal-157 → βAla-156/βThr-157) were constructed, and their properties were studied extensively. The βSer-156 mutant was active in ATP synthesis and had ~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 (βAla- 155, βSer-155, βAla-156, and βCys-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 βAla-156 and βCys-156 enzymes and 102-fold lower with the βAla-155 and βSer-155 enzymes. The βThr-156 → Ala or Cys enzyme showed an altered response to Mg2+, suggesting that βThr-156 may be closely related to Mg2+ binding. These results suggest that βLys-155 and βThr-156 are essential for catalysis and are possibly located in the catalytic site, although βThr-156 could be replaced by a serine residue.
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M3 - Article
C2 - 1400377
AN - SCOPUS:0026700075
VL - 267
SP - 20571
EP - 20576
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -