Variants of subtilisin BPN' with altered specificity profiles.
A strategy for increasing the size of the S4 binding pocket was used to improve the specificity of subtilisin BPN' toward substrates with large hydrophobic P4 side chains. This approach involves single and double amino acid replacements at positions 104, 107, and 126. Previously, alteration of I107 to glycine has been found to increase the specificity of subtilisin toward leucine, isoleucine, and phenylalanine as P4 residues by up to 214-fold. Replacement of Y104 by alanine also yields a similar improvement in specificity. However, this subtilisin variant favors isoleucine and phenylalanine over leucine. When L126 was replaced by valine, alanine, and glycine, respectively, only the L126A subtilisin variant, which possesses a 28-fold-increased catalytic efficiency for isoleucine compared with all other substrates tested, showed a significantly improved specificity profile. As inferred from the double-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of the effects of the single amino acid replacements on the kinetic parameters are additive. The I107G/L126V mutant subtilisin has the largest improvement in P4 substrate specificity reported so far: kcat/KM is increased 340-fold for leucine compared to alanine. By contrast, the specificity profile of the I107G/Y104A mutant enzyme is impaired in comparison with that of the corresponding single mutants. Therefore, the design of high-specificity subtilisin variants through the combination of single amino acid replacements in the S4 pocket appears to be nontrivial due to the interference of the introduced structural changes.
Rheinnecker
M
M
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.
Eder
J
J
Pandey
P S
PS
Fersht
A R
AR
D000595:Amino Acid Sequence; D001665:Binding Sites; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D013381:Subtilisins