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.
Department of Bioscience, Fukui Prefectural University, Yoshida-gun, Japan. hiro@fpu.ac.jp
H
H
Takagi
T
T
Maeda
I
I
Ohtsu
Y C
YC
Tsai
S
S
Nakamori
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