Effect of Divalent Cations on Bovine Pancreatic Ribonucrease*

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Abstract
1. Effect of various divalent cations on Ribonuclease A [EC 2.7.7.16, ribonucleate pyrimidinenucleotido-2′transferase (cyclizing)] (RNase A) activity was reinvestigated with well-characterized substrates such as benzyl cytidine 3′phosphate (C-3′p-Bz), cytidine 2', 3′cyclic phosphate (C-cyclic-p) at pH 7.0, μ 0.1 and 37°C. At concentrations of 10−3 and 10−4 M, Cu++ ion, and to a lesser extent Zn++ and Hg++ ions had an inhibitory effect. Other ions such as Mg++, Ca++ and Mn++ about which there have been reports of diverse results were found to have little effect. 2. The observed inhibition of RNase A activity at a low concentration of Cu++ and Zn++ at the initial stage of hydrolysis of C-3′p-Bz cannot satisfactorily be explained by the formation of a ternary complex [E-Me++-cytidine 3′phosphate (C-3′p)] suggested by Ross et al. A possible mechanism of the inhibition in the early stage of the reaction seems to be due to the formation of [E-Me++] complex. In the later stage of the reaction when the product, C-3′p, accumulates to some extent, the formation of a ternary complex [E-Me++-C-3′p] might be a factor for the inhibition. 3. Metal ions such as Cu++ and Zn++ are non-competitive inhibitors for RNase A. 4. A cluster including the active center of the enzyme appears to constitute the metal binding site. Even when any one of amino acid residues in the active center is modified, the cluster can still combine metal ions, but it seems to lose the binding ability with metal ions when two histidine residues in the active site are modified. 5. Cytidine 2′phosphate (C-2′p) as well as C-3′p is found to form the ternary complex [E-Me++-CMP] by the results of equilibrium dialysis and spectrophotometry. 6. The effect of temperature and ionic strength on the metal binding of the enzyme was studied.