Enzyme-catalyzed formation of semisynthetic staphylococcal nuclease using a new synthetic fragment, [48-glycine]synthetic-(6-49).

While trypsin can catalyze resynthesis of the peptide bond between fragments in the noncovalent complex of nuclease-T-(6-48) and nuclease-T-(49-149), this reaction leads to excision of Lys 49 and formation of inactive [des Lys 49]-nuclease-(6-149). To provide a method for making active active covalent semisynthetic nuclease, we chemically synthesized the fragment of residues 6 to 49 in which lysine 48 was replaced by glycine. This peptide was made using the recently described solid phase support, 4-(oxymethyl)phenylacetamidomethyl-polystyrene. The resultant crude polypeptide exhibited 30-50% of native nuclease-T enzymatic activity when added to native nuclease-T-(50-149). When the non-covalent complex formed by native nuclease-T-(50-149) and a 10-fold molar excess of [Gly 48]synthetic-(6-49) was equilibrated with trypsin in 90% glycerol, an increase in enzymatic activity from 8 to 32% (versus nuclease) was observed. Simultaneously, approximately 20% conversion of nuclease-T-(50-149) to nuclease-molecular weight material was observed by gel electrophoretic analysis. These data indicate that a covalent semisynthetic species is formed with activity about equal to that of native nuclease. The results confirm the importance of loop integrity on catalytic site organization. The Gly-48-containing fragment system defined above can allow preparation of semisynthetic nuclease sequence analogs.