Effect of Various Nucleotides on Folding and Enzymic Properties of a Synthetic 63‐Residue Analog of Ribonuclease A and Natural Ribonuclease A

When a reduced synthetic 63‐residue analog of ribonuclease A (RNase A) was reoxidized in the presence of 2′‐CMP, a competitive inhibitor of RNase A, the overall activity after removal of the inhibitor was lower than that of the same analog reoxidized in the absence of 2′‐CMP. However, reoxidation in the presence of 2′‐CMP diminished the activity toward poly(U) more strongly than that toward poly(C). To investigate the influence of substrate analogs on folding and enzymic properties of the natural enzyme, reduced RNase A was reoxidized in the presence of 3′‐CMP, 3′‐UMP, adenosine 2′:3′‐monophosphate (2′:3′‐AMP) or guanosine 2′:3′‐monophosphate (2′:3′‐GMP). The activities of ribonuclease reoxidized in the presence of 3′‐CMP were essentially unaltered whereas RNase A refolded in the presence of 3′‐UMP had 150% activity toward 2′:3′‐UMP and RNase A refolded in the presence of 2′:3′‐AMP had 170% activity toward poly(A) as compared with the activity of untreated natural RNase A against these substrates. Kinetic studies using uridine 2′:3′‐monophosphate (2′:3′‐UMP) as substrate showed that the maximum velocity of the reaction catalyzed by RNase A which had been reoxidized in the presence of 3′‐UMP was twice that found with untreated native RNase A as enzyme. Comparison of the Michaelis constants indicated that refolding of RNase A in the presence of 3′‐UMP did not increase the affinity for 2′:3′‐UMP. The conformational isomers of RNase A formed through reoxidation in the presence of various nucleotides were not very stable. Thus, raising the temperature to 40°C lowered the activity against poly(A) from 170% to 115%. However, it cannot be excluded that conformational isomers of this kind are also formed during the biosynthesis of RNase A.