Identification of Trp300 as an important residue for Escherichia coli leader peptidase activity.
We previously reported that leader peptidase from Escherichia coli was extensively inactivated by reaction with N-bromosuccinimide with concomitant and selective modification of the Trp300 and Trp310 residues [Kim, Y.-T., Muramatsu, T. & Takahashi, K. (1995) J. Biochem. (Tokyo) 117, 535-544]. This indicated that one or both of these tryptophan residues are important for the activity of the enzyme. In order to define further the role of individual tryptophan residues in the activity of leader peptidase, site-directed mutagenesis studies were performed to replace each tryptophan residue with phenylalanine and/or alanine. The replacements of Trp20, Trp59, Trp261, Trp284, and Trp310 with phenylalanine hardly affected the enzyme activity toward a synthetic peptide substrate and the ability to complement the temperature sensitivity of the mutant leader peptidase in E. coli IT41. In contrast, the activity toward the synthetic substrate was significantly decreased by replacement of Trp300 with phenylalanine or alanine. The kcat values of the W300F and W300A mutant enzymes were reduced to 42% and 22%, respectively, of that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type enzyme. Moreover, the complementing ability in E. coli IT41 was lost (almost) completely when Trp300 was replaced with phenylalanine or alanine. These results strongly indicate that Trp300 in leader peptidase is important for the catalytic mechanism and/or the construction of the active site structure of the enzyme.
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Y T
YT
Kim
T
T
Muramatsu
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K
Takahashi
D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D012697:Serine Endopeptidases; D014364:Tryptophan / Q000378:metabolism*
D008565:Membrane Proteins; D014364:Tryptophan; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase