Remarkable improvements of a neutral protease activity and stability share the same structural origins

Stabilizing an enzyme while improving its activity may be difficult with respect to a general trade off relation between stability and function at the level of individual mutations. We have used site-directed mutagenesis to investigate the possibility of parallel improvements of thermostability and activity in the neutral protease from Salinovibrio proteolyticus. Four out of seven point mutations are able to promote both activity and thermostability individually and combinedly. The catalytic efficiency (k(cat)/K(m)) of four-amino acid substituted variant (quadruple mutant) at 60 degrees C is 18-fold higher than wild type, whereas at optimum temperature is almost 50-fold higher. Quadruple mutant shows an upward shift of 14 degrees C in the temperature optimum, and a 20-, 24-, 7- and 5-fold increase in half-life at 60, 65, 70 and 75 degrees C, respectively, as a result of enhanced calcium binding. Theoretical studies have provided evidences that hinge-bending angle is reduced by amino acid substitutions. Finally, we conclude that the extended surface region between residues 187-228, which involves three out of four beneficial mutations, influences the hinge angle which is determinant for catalysis and also involves the structural calcium which is critical for stability.