Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase: Histidine 142α is a facilitative catalytic residue

G. X. Luo, J. S. Nishimura

Resultado de la investigación: Articlerevisión exhaustiva

4 Citas (Scopus)

Resumen

There are 11 histidine residues in Escherichia coli succinyl-CoA synthetase. His-246α is well established as the phosphorylation site of the enzyme. Replacement of this histidine by asparagine (Mann, C. J., Mitchell, T., and Nishimura, J. S. (1991) Biochemistry 30, 1497-1503) or by aspartic acid (Majumdar, R., Guest, J. R., and Bridger, W. A. (1991) Biochim. Biophys. Acta 1076, 86-90) through site-directed mutagenesis resulted in complete loss of enzyme activity. Chemical modification experiments suggested a second histidine at the active site (Collier, G. E., and Nishimura, J. S. (1979) J. Biol. Chem. 254, 10925-10930). In the present study, we have changed His-142α to an asparagine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is practically devoid of enzyme activity but can be thiophosphorylated with adenosine 5'-O-(thiotriphosphate) and dethiophosphorylated with ADP at rates that are significantly faster than those with wild type enzyme. The observation that phosphorylated mutant enzyme can be dephosphorylated with succinate and with succinate plus desulfo-CoA at rates comparable with those with wild type enzyme suggests that mutant enzyme can bind succinate and CoA. Dethiophosphorylation of the enzyme in the presence of CoA plus succinate proceeds much faster with wild type than with mutant. While there was no significant change in K(CoA) or K(succinate), the turnover number for dethiophosphorylation of the mutant was 10-fold lower. These data are consistent with location of His-142α at the active site and a facilitative role for this residue in catalysis.

Idioma originalEnglish (US)
Páginas (desde-hasta)20781-20785
Número de páginas5
PublicaciónJournal of Biological Chemistry
Volumen266
N.º31
EstadoPublished - 1991
Publicado de forma externa

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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