A strategy for increasing the size of the S4 binding pocket was used t
o improve the specificity of subtilisin BPN' toward substrates with la
rge hydrophobic P4 side chains. This approach involves single and doub
le amino acid replacements at positions 104,107, and 126. Previously,
alteration of I107 to glycine has been found to increase the specifici
ty of subtilisin toward leucine, isoleucine, and phenylalanine as P4 r
esidues 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 repla
ced by valine, alanine, and glycine, respectively, only the L126A subt
ilisin variant, which possesses a 28-fold-increased catalytic efficien
cy for isoleucine compared with all other substrates tested, showed a
significantly improved specificity profile. As inferred from the doubl
e-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of th
e effects of the single amino acid replacements on the kinetic paramet
ers are additive. The I107G/L126V mutant subtilisin has the largest im
provement in P4 substrate specificity reported so far: k(cat)/K(M) is
increased 340-fold for leucine compared to alanine. By contrast, the s
pecificity profile of the I107G/Y104A mutant enzyme is impaired in com
parison 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 nont
rivial due to the interference of the introduced structural changes.