PURIFICATION AND CHARACTERIZATION OF S1 MUTANTS OF ALPHA-LYTIC PROTEASE HAVING ALTERED CATALYTIC PROPERTIES

A procedure is described for purifying alpha-lytic protease and its mu tants from culture supernatants of recombinant Escherichia coli. The m ethod affords substantial amounts (approx. 80 mg) of homogeneous enzym e. We compared the cleavage preferences of wild-type alpha-lytic prote ase and of mutants containing the substitutions Ala190 (''parent''), A la190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9) , and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found b road agreement between the results obtained with synthetic ester and a mide substrates. Kinetic constants were determined for the purified en zymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of k(cat)/K-m it cl eaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wi ld-type protease. The Ala190/His213 enzymes showed a preference for cl eavage at His and Met residues. Not only were their k(cat) values for cleavage at His increased (in relation to the Ala190 enzyme) by an ord er of magnitude, but they also exhibited large decreases in k(cat)/K-m for cleavage at other residues; for example, the value for cleavage a t Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-I I fusion protein at a unique His residue and also at a nearby Asn resi due. (C) 1994 Academic Press, Inc.