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Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue
Author(s) -
Takagi Hiroshi,
Maeda Tadao,
Ohtsu Iwao,
Tsai Yeng-Chieh,
Nakamori Shigeru
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)01014-9
Subject(s) - subtilisin , residue (chemistry) , periplasmic space , bacillus subtilis , side chain , steric effects , chemistry , stereochemistry , biochemistry , glycine , amino acid , escherichia coli , mutant , biology , enzyme , bacteria , organic chemistry , genetics , gene , polymer
Substitution of the conserved Gly 127 for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis . The crystallographic findings suggested that Gly 127 is responsible for accepting even the large P1 substrates, and the marked change of specificity was attributed to the introduction of a side chain in this position. To test this hypothesis, Gly 127 was replaced with 3 non‐charged amino acids, Ala, Ser and Val. When assayed with synthetic peptide substrates, all mutants purified from the periplasmic space in Escherichia coli showed a marked preference for small P1 substrate up to 150‐fold relative to the wild‐type. The kinetic data and molecular modeling analysis suggest that large hydrophobic P1 residues were unable to access the binding pocket due to steric hindrance.