title: Engineering a novel specificity in subtilisin BPN'.
abstract: The specificity of subtilisin BPN' toward substrates with large hydrophobic P4 residues has been improved by single amino acid replacements at positions 104 and 107. Mutations were designed to (i) increase the size of the P4 binding pocket by replacing Ile107, which is at the bottom of the S4 pocket, by Val, Ala, and Gly and (ii) lose the hydrogen bond between Tyr104 and Ser130 at the entrance of the P4 binding pocket by changing Tyr104 to Phe and thus reduce interactions between small P4 side chains and residue 104. All mutant subtilisins, except for I107V, have increased specificity for residues with large side chains at P4 compared with wild type. Using the conventional definition of specificity as the competition of different substrates for the same enzyme, the I107G mutant subtilisin has one of the largest improvements in substrate specificity reported for subtilisin so far; kcat/KM is increased > 200-fold for Phe compared with Ala as the P4 residue. Further, the activity of I107G toward its specific substrate is comparable to that of the wild-type enzyme. Surprisingly, much of the increase in specificity on mutation of Ile107-->Gly appears to result from a lesion that is transmitted through the structure and effects catalysis. The value of kcat/KM for the small substrate acetyltyrosine ethyl ester, which binds to the S1 pocket, drops by 93% on mutation of Ile107-->Gly. The lesion in subtilisin I107G is complemented, however, on binding of longer substrates that have a large hydrophobic P4 amino acid side chain that can bind in the S4 pocket.
authors.affiliation: MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Rheinnecker
authors.forename: G
authors.initials: G
authors.lastname: Baker
authors.forename: J
authors.initials: J
authors.lastname: Eder
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000138:chemical synthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; D013381:Subtilisins