ENGINEERING SUBTILISIN YAB - RESTRICTION OF SUBSTRATE-SPECIFICITY BY THE SUBSTITUTION OF GLY124 AND GLY151 WITH ALA

The 3-D structure of subtilisin YaB was computer modelled using the st ructures of subtilisin BPN', subtilisin Carlsberg and thermitase as te mplates. Gly124 and Gly151 located on both sides of the waist of the S 1 pocket were selected for site-directed mutagenesis based on the mode lled structure. The mutated ale genes coding for the mutant subtilisin YaB were expressed in Bacillus subtilis DB104., All of the G124 and G 151 series of mutants exhibited an increase of relative catalytic acti vity for elastin-orcein against casein and myofibrillar proteins. The S1 substrate specificity of G124A, G124V and G151A mutants were assess ed using various carbobenzoxy-amino acid-nitrophenyl esters and succin yl-Ala-Ala-(Pro or Val)(Ala, Phe or Leu)-p-nitroanilide [AA(PN) (A/F/L )], While G124A and G124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzed Ala, Leu and Gly esters. The G124A and G124V m utants did not hydrolyze AAPF and AAPL., However, these two mutants hy drolyzed AAPA and AAVA with k(cat)/K-m values approximately 3-10-fold higher than those of the wild-type enzyme. The G151A mutant did not hy drolyze AAPF, but hydrolyzed AAPL, AAPA and AAVA with k(cat)/K-m value s approximately l-il-fold higher than those of the wild-type enzyme. T hese results clearly indicate that the S1 substrate specificity of G12 4A and G124V mutants was restricted to Ala and Gly, and G151A mutant t o Ala, Gly and Leu.