INHIBITION AND SUBSTRATE COMPETITION KINETICS IN ANALYSIS OF PORCINE THYROID ALKALINE RIBONUCLEASES SPECIFICITY TOWARD SYNTHETIC RNAS AND TRANSFER-RNA

Inhibition and substrate competition kinetics demonstrated that tRNA i s a highly preferred substrate of thyroid alkaline RNase. The pyrimidi ne-specific RNase cleaved poly(C) 2.8 x 10(5) faster than poly(U). k(c at):K-M ratios for tRNA and poly(C) based on molecular weights failed to predict preference when both were present. Competition experiments between poly(C) and tRNA revealed tRNA was a tight-binding competing s ubstrate and the cytidylate residues in the 3'-CCA terminus of tRNA we re preferred about 280:1 over those in poly(C). Poly(U) was competitiv e with tRNA. When poly(C) was the substrate, inhibition type by poly(G ) depended on poly(G) concentration. Neither tRNA lacking its 3' termi nal cytidylyl(3'-5')adenosine and terminating in a 2':3' cCMP residue, tRNA lacking its 3/terminal 5'AMP residue, guanosine, nor guanylyl(3' -5')guanylyl(3'-5')guanosine were inhibitors. Product inhibition by ad enosine and 2':3' cCMP showed the kinetic mechanism for cleavage of tR NA was ordered uni bi.