A Comparative Study on Enzymatic Activity of Bovine Pancreatic Ribonuclease A, Ribonuclease S and Ribonuclease S*

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Abstract
The enzymatic activity of subtilisin-produced derivatives of ribonuclease A (RNase A) [EG 2. 7. 7. 16, ribonucleate pyrimidinenuclcotido2′-transferase (cyclizing)], ribonuclease S (RNase S) and ribonuclease S (RNase S), has been compared with that of the original enzyme using RNA and synthetic low-molecular weight nucleotide derivatives as substrates at varying pH's and temperatures. 1. Whenever low-molecular weight compounds were used as substrates, pH optimum of each of the modified enzymes for both the steps of cyclization and hydrolysis shifted to more neutral side than that of the original enzyme. The pH optimum at 16°G was 6.5 for RNase S, 5.5 for RNase A and between 6.5 and 5.5 for RNase S. The shifts in pH optimum of both the modified enzymes at 37°C were less remarkable than those at 16°C. 2. When RNA was used as substrate, three RNases showed an identical pH optimum of 8.0 at 16°G as well as 37°C. 3. Specific activities of the three enzymes increased in an order of RNase ASS at 16°C and RNase SSA at 37°G irrespective of substrates used. This opposite order of the specific activities at 37°G against at 16°C is probably due to the differences in heat sensitivity of the three enzymes. 4. Kinetic parameters at 15°C, pH 7.0 were estimated using cytidine 2′, 3′-cyclic phosphate (C-cyclic-p), uridine 2′, 3′-cyclic phosphate (U-cyclic-p) or eight kinds of di-ribonucleoside monophosphate (XpY) as substrate. The values of Vmax for the three enzymes increased in the following order: RNase ASS; the values of Km exhibited, with a few exceptions, the following tendency: RNase A≤RNasc SS. 5. The marked accelerating effect of Y bases on the hydrolysis of XpY's proposed by Witzel and Barnard for the reaction of RNase A was also observed in the reactions catalized by subtilisin-modified RNases. From the different accelerating effects of Y bases observed with each enzyme, the possibility of direct interaction of Y bases with the enzymes was discussed.

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