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.
Highlights
Nuclease-(1-126), containing 89%of the front comer of the model (4-6)
To at- We have further investigated the conformational properties tempt to learn whetheor r not the active population of nuclease-(1-126) has the nativeconformation, we have examined nuclease-(1-126) with respect to enzymatic kinetics with and without the competitive inhibitor deoxythymidine3’,5’-diphosphate, effecotf temperature on enzymatic activity, binding of pdTp in the of nuclease-(1-126), in relation to intrinsic enzymatic activity
Formation or stabilization of ence of Ca2+with an apparent association constant approxithe active site of nuclease-(1-126) by binding with li- mateIy three orders of magnitude smaller thanthat with gands is not associated with cooperative folding of the nuclease A and that the conformation of liganded nucleaseentire polypeptide chain
Summary
Homogeneity of the Nuclease-(l-l26) Preparation-The purified nuclease-(1-126) was homogeneous as ju d ged by a symmetrical peak obtained inthe elution profile bygel fitration (see the legend to Fig. 1). There has The enzymatic kinetic parameters ofnuclease-(l-126), nuclease A, beennoextensivezonalspreading or deformation of the symmetrical peak in the elution profile A t p H 8.8and 24 f 1“ C mer-dimer (or polymer)equilibrium) in the concentration range from 1 to 6 mg per ml of nuclease-(1-126), a n d the Enzymatic activity was measured a t pH 8.8 at 24 f 1“C according to themethod of Cuatrecasas et al (13) as described in the legend to absorbance at 280 nm obeys Lambert-Beer law for solutions Fig..
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