ENERGETICS OF CATALYSIS BY RIBONUCLEASES - FATE OF THE 2',3'-CYCLIC PHOSPHODIESTER INTERMEDIATE

Ribonucleases catalyze the hydrolysis of the P-O-5' bond in RNA. This reaction occurs in two steps: transphosphorylation of RNA to a 2',3'-c yclic phosphodiester intermediate and hydrolysis of this intermediate to a 3'-phosphomonoester. P-31 NMR spectroscopy was used to monitor th e accumulation of the 2',3'-cyclic phosphodiester intermediate during the transphosphorylation and hydrolysis reactions catalyzed by various ribonucleases and by small molecules. The intermediate was found to a ccumulate during catalysis by monomeric bovine pancreatic ribonuclease A (RNase A), a dimer and a trimer of RNase A, bovine seminal ribonucl ease, RNase T-1, barnase, and RNase I. These enzymes, which are of wid ely disparate phylogenetic origin, released rather than hydrolyzed mos t of the intermediate formed by transphosphorylation of RNA. In contra st, the intermediate did not accumulate during catalysis by hydroxide ion or imidazole buffer. In the presence of these small molecules, hyd rolysis is faster than transphosphorylation. A trapping experiment was used to assess the throughput of the reaction catalyzed by RNase A. [ 5,6-H-3]Uridylyl-(3'-->5')adenosine was incubated with RNase A in the presence of excess unlabeled uridine 2',3'-cyclic phosphodiester, whic h dilutes the specific radioactivity of any released cyclic intermedia te. Only 0.1% of the RNA substrate was found to be both transphosphory lated and hydrolyzed without dissociating from the enzyme. These resul ts suggest that ribonucleases have evolved primarily to catalyze RNA t ransphosphorylation and not RNA hydrolysis.