The mechanism of stabilization of the structure of nuclease-T by binding of ligands.
The rate of unfolding of Nuclease-T at pH 8,20 degrees was determined as a function of concentration of the ligands deoxythymidine 3',5'-diphosphate (pdTp) and Ca2+ on the basis of the rate of exchange between free fragment, Nuclease-T(50-149) and labeled fragment, Nuclease-T-(50-149) incorporated in the structure of nuclease-T (Taniuchi, H. (1973) J. Biol. Chem. 248, 5164-5174). The rate constant of unfolding of unliganded Nuclease-T' was 4.6 times 10-4s-1. Those of Nuclease-T' bound with pdTp, with Ca2+, and with both pdtp and Ca2+ were 9.0 times 10-5, 1.6 times 10-4, and 2.2 times 10-5s-1, respectively. The association constants of pdTp and Ca2+ with Nuclease-T' were found to be 1.0 times 10-4 and 2.0 times 10-2 m-1, respectively. Those of pdTp with Nuclease-T' plus Ca2+ and of Ca2+ with  Nuclease-T'  plus pdTp were 4 times 10-5 and 1.4 times 10-4M-1, respectively. The calculation of free energy change on the basis of the association constants shows that the magnitude of negative free energy change involved in the binding of either of the two ligands increases by approximately 2 kcal when the other ligand is already bound. There is a correlation between the free energy change and the specifically coupled with the cooperative interacions operating throught the three-dimensional structure resulting in strengthening of the interactions throughtout the structure, including those with the ligands, without a large change in conformation.
D002118:Calcium; D006358:Hot Temperature; D008024:Ligands; D008433:Mathematics; D009696:Nucleic Acids; D010727:Phosphoric Diester Hydrolases; D011485:Protein Binding; D011487:Protein Conformation; D013210:Staphylococcus / Q000201:enzymology; D013816:Thermodynamics; D013942:Thymine Nucleotides
D008024:Ligands; D009696:Nucleic Acids; D013942:Thymine Nucleotides; D010727:Phosphoric Diester Hydrolases; D002118:Calcium