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&prim;e: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. [ABSTRACT FROM AUTHOR]