Differences in the specificities of the highly alkalophilic proteinases Savinase and Esperase imposed by changes in the rigidity and geometry of the substrate binding sites

Savinase and Esperase are closely related highly alkalophilic proteinases p roduced by Bacillus lentus. They are suitable couple for investigating the structural basis of proteinase specificity due to the identity of the catal ytic and the differences in the substrate binding sites. Two of the substit utions in these sites are very important: T129P and G131P. The two prolines provide an extra rigidity of the Savinase-binding site. The substitutions S166N and Q191T in the S1 recognition loop change the binding geometry of t he substrate P1 residue. The geometry of S1 in Esperase is more favorable f or binding and catalysis in comparison to that in Savinase. Differences in P3 specificity are probably created by the substitution V104L, which influe nces the conformation of S3. Leu in position 104 is more favorable for the binding of Phe to S4 than Val, The lower affinity and catalytic efficiency as well as more narrow proteolytic specificity of Savinase in comparison to those of Esperase are explained with the extra rigidity and unfavorable ch anges in geometry of the substrate binding site of the first enzyme. (C) 20 01 Academic Press.