pH-Profile of the Kinetic Parameters of Ribonuclease T1

Abstract

1. Kinetic parameters, K_m and V_max, of RNase T₁ [EC 2.7.7.26] were obtained using G-cyclic-p as substrate at various pH's. From the _pK_m-, log V_max-pH profile, two _pK_a's of free enzyme (_pK_e) and _pK_a of enzyme-substrate complex (_pK_es) were estimated (_pK_e 5.7 and about 7.5; _pK_es 3.7, 6.7 and about 7.4). 2. From the _pK_t-pH. profile of RNase T₁ using G-2′-p as competitive inhibitor, the _pK_e values of 5.7 and about 7.5 were also obtained. 3. Based on the values obtained above, the possibility of the presence of histidine residues in the active site of the enzyme was discussed. 4. From the _pK_t- and _pK_m-pH profile, the preferable binding form of G-2′-p with the enzyme was presumed to be monoanionic species.