Structure-Function Relationships at the Active Site of Nuclease-T

Abstract

Abstract Solid phase peptide synthesis has been used to study structural requirements at the active site of staphylococcal nuclease-T', the noncovalent complex of the polypeptides nuclease-T-(6-48) and nuclease-T-(49-149) (residues 6 through 48 and 49 through 149 of native nuclease, respectively). In light of the importance of glutamic acid 43 in nuclease-T' catalysis (Chaiken, I. M., and Sanchez, G. R. (1972) J. Biol. Chem. 247, 6743–747), analogues of nuclease-T-(6-48) with progressively longer deletions at the COOH terminus have been synthesized, and their binding to native nuclease-T-(49-149) to produce an active complex has been investigated. The results indicate that residues glutamic acid 43, threonine 44, lysine 45, histidine 46, proline 47, and lysine 48 are not necessary for formation of a nuclease-T'-like complex. On the other hand, threonine 44 is critical for normal enzymic activity. The effect of threonine 44 appears to be due to its contribution to the peptide bond with glutamic acid 43 and not to its specific side chain moiety. The synthesis of an analogue of nuclease-T-(6-48) with alanine at position 19 in place of aspartic acid was also undertaken. No enzymic activity is generated by this peptide in the presence of nuclease-T-(49-149), although complex formation appears to occur. This finding is consistent with the view, based on the crystal structure of the nuclease-Ca++-deoxythymidine 3',5'-diphosphate complex (Cotton, F. A., Bier, C. J., Day, V. W., Hazen, E. E., Jr., and Larsen, S. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 243–249), that the side chain carboxyl group of aspartic acid 19, along with those of aspartic acid residues 21 and 40 and glutamic acid 43, participates in the binding of the essential calcium ion.