A Study of the Role of Histidine Residue 46 in Staphylococcal Nuclease by Solid Phase Peptide Synthesis

Synthetic polypeptides corresponding to Residues 6 to 47 and 9 to 47 of staphylococcal nuclease, as well as these same sequences containing glycine in place of the normally occurring histidine residue at position 46 (6–47, Gly46 and 9–47, Gly46), were prepared by the Merrifield solid phase method. The normal histidine-containing sequences and the Gly46 analogues generated similar levels of DNase and RNase activities when mixed with an equimolar amount of native peptide containing Residues 49 or 50 to 149 (P3). The activities for all synthetic peptides, in the crude state, were about 4 to 5% of that given by the noncovalent complex (native nuclease-T) formed by an equimolar mixture of native P2 (Residues 6 to 48) with native P3. The active, semisynthetic nuclease-T species for both analogue and normal peptides were similar not only in activity but in resistance to trypsin digestion and in fluorescence emission characteristics. The semisynthetic nuclease-T species generated by both 6–47 and 6–47, Gly46 were purified, after trypsin digestion in the presence of deoxythymidine 3',5'-diphosphate and Ca++, by phosphocellulose chromatography. Both forms, after such purification, exhibited about 50 to 60% of the DNase activity of native nuclease-T. The results demonstrate that histidine Residue 46 is not essential for either enzyme activity or for certain other properties characteristic of nuclease and nuclease-T.