A kinetic study has been made of the ribonuclease-catalyzed hydrolyses of three cyclic nucleotides, cytidine-2′,3′-phosphate, uridine-2′,3′-phosphate, and N6,O5′-diacetyl cytidine-2′,3′-phosphate. Rates were measured at pH values ranging from 6 to 8.5. The variation of the kinetic parameters with pH showed that the free enzyme possesses two active groups, having pK values of 5.4 and 7.25. When the enzyme–substrate complex is formed, the pK values of the groups are increased to 6.6 and 8.4. The pK values identify these groups as imidazole groups and show that two histidine residues are present at the active site. Since both increase in pK on complex formation, it is concluded that the acid imidazole group binds the substrate, but that the basic imidazole group cannot be concerned in substrate binding and must function only in the hydrolytic step. The results indicate that the pyrimidine base is concerned in the hydrolytic step and not solely in binding, as had been postulated. It is concluded from all of the evidence that four specific sites are present at the active center of the enzyme; three are involved in binding and one in catalysis. It is proposed that the active site of ribonuclease is composed of: the histidine residue in position 12, which catalyzes the hydrolytic step; the histidine residue in position 119, which binds the 2′-ribose oxygen atom in the substrate; the lysine residue in position 41, which binds the phosphate group or anion; and the aspartic acid residue in position 121, which binds the nitrogen atom at N1 in the pyrimidine base. A mechanism for enzyme–substrate complex formation and subsequent hydrolysis is proposed.
Association-dissociation equations, distinct from Michaelis-Menten equation for the quantification of the net flux of reactants with or without immobiliser.
