Inhibition and Substrate Competition Kinetics in Analysis of Porcine Thyroid Alkaline Ribonuclease's Specificity Toward Synthetic Rna's and Trna

Abstract

Inhibition and substrate competition kinetics demonstrated that tRNA is a highly preferred substrate of thyroid alkaline RNase. The pyrimidine-specific RNase cleaved poly(C) 2.8 × 10⁵ faster than poly(U). k_cat: 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 substrate and the cytidylate residues in the 3prime;-CCA terminus of tRNA were preferred about 280: 1 over those in poly(C). Poly(U) was competitive with tRNA. When poly(C) was the substrate, inhibition type by poly(G) depended on poly(G) concentration. Neither tRNA lacking its 3prime; terminal cytidylyl(3prime;-5prime;)adenosine and terminating in a 2prime;:3prime; cCMP residue, tRNA lacking its 3prime; terminal 5prime;AMP residue, guanosine, nor guanylyl(3prime;-5prime;)guanylyl(3prime;-5prime;)guanosine were inhibitors. Product inhibition by adenosine and 2prime;:3prime; cCMP showed the kinetic mechanism for cleavage of tRNA was ordered uni bi.