Further study of the conformation of nuclease-(1-126) in relation to intrinsic enzymatic activity

Abstract

Nuclease-(1-126), although containing 89% of the amino acid sequence which folds to the ordered structure of nuclease A, is disordered and highly flexible (Taniuchi, H., and Anfinsen, C. B. (1969) J. Biol. Chem. 243, 4778-4786). On the other hand, Sachs et al. (Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfinsen, C. B. (1974) Nature 251, 242-244) have demonstrated intrinsic enzymatic activity for nuclease-(1-126). To attempt to learn whether or not the active population of nuclease-(1-126) has the native conformation, we have examined nuclease-(1-126) with respect to enzymatic kinetics with and without the competitive inhibitor deoxythymidine 3',5'-diphosphate (pdTp), effect of temperature on enzymatic activity, binding of pdTp in the presence of Ca2+ and intrinsic viscosity, Stokes radius, CD, and response to trypsin action in the presence and absence of pdTp and Ca2+. The results indicate that the conformation of nuclease-(1-126) bound with pdTp in the presence of Ca2+ is partially constrained but still highly flexible below 30 degrees C, outside the range of thermal transition exhibited by the ordered elements of nuclease-(1-126). Thus, formation or stabilization of active site of nuclease-(1-126) by binding with ligands is not associated with cooperative folding of the entire polypeptide chain. Considering that nuclease-(1-126) does not bind to nuclease-(127-149) but does to nuclease-(111-149), the results are consistent with the idea that the specific cooperative interactions, providing extra stabilizing energy required for maintaining the polypeptide chain in the ordered state of nuclease A, may be disrupted for nuclease-(1-126) primarily due to cleavage of the peptide bond between residues 126 and 127. Then, it may be thought that binding with ligands does not compensate for this disruption.