Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue.
Substitution of the conserved Gly127 for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis. The crystallographic findings suggested that Gly127 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, Gly127 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.
Takagi
H
H
Department of Bioscience, Fukui Prefectural University, Yoshida-gun, Japan. hiro@fpu.ac.jp
Maeda
T
T
Ohtsu
I
I
Tsai
Y C
YC
Nakamori
S
S
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D005998:Glycine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D013381:Subtilisins; D005998:Glycine