Ribonuclease and Oligoribonucleotide Synthesis

The nucleotides, 5'-O-phosphoryl uridine 2',3'-cyclic phosphate and 5'-O-methylphosphoryl uridine 2',3'-cyclic phosphate, and the dinucleotides obtained from the reaction of these compounds with uridine have been synthesized and characterized. The 5'-substituted nucleotides were prepared to serve as nonpolymerizing substrates in kinetic studies of oligoribonucleotide synthesis. Reaction conditions were obtained which allowed a comparison of the synthetic activity of the native enzyme and the synthetically active derivative, e-dinitrophenyllysine-41-ribonuclease. Irrespective of substrate, the synthetic activity of the dinitrophenylated enzyme differed from that of unmodified ribonuclease with regard to optimal pH, proportionality to enzyme concentration, reaction velocity, and the effect of substrate concentration. In reactions catalyzed by ribonuclease A, the kinetic parameters (Km and Vm) for synthetic activity were identical with either nonpolymerizing pyrimidine cyclic phosphate. The e-dinitrophenyllysine-41-ribonuclease-catalyzed reactions yielded kinetic values which varied with the 5'-substituent of the cyclic phosphate. Relative to native ribonuclease, the apparent affinity (Km) of the substrates for the modified enzyme was not diminished, although the reaction velocity (Vm) was reduced. Analysis of the data suggests that the lysine-41 residue is not essential for pyrimidine cyclic phosphate binding, but may participate in the catalytic process required for synthetic activity.