Reactions at the 3′ Terminus of Transfer Ribonucleic Acid: III. CATALYTIC PROPERTIES OF TWO PURIFIED RABBIT LIVER TRANSFER RIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASES

Abstract

Abstract The catalytic properties of two purified rabbit liver tRNA nucleotidyltransferases were examined and compared. Both enzymes incorporated AMP, CMP, and UMP into tRNA, but were inactive with GTP or dATP. The pH optima for the various nucleotides and for both enzymes were similar and were in the range of pH 9 to 10. Differences were observed in the response of the two enzymes to the assay temperature, such that the activation energies for phosphocellulose Peak II were about 50% higher than for Peak I. The apparent Km values for nucleoside triphosphates were essentially identical for both enzymes. For ATP with tRNApCpC as cosubstrate the Km values were 3 mm and 2 mm for enzymes I and II, respectively; using tRNApC as cosubstrate the apparent Km with Peak II was 2 mm. In the case of CTP, biphasic double reciprocal plots were found which suggested the existence of two CTP binding sites. The data for each enzyme could be fit to curves generated from Km values of 0.004 mm and 0.4 mm and appropriate Vmax values. Similar Km values for CTP were obtained with tRNApC or tRNApX as cosubstrate although the Vmax values differed. UTP, which acted as an analogue of CTP, gave apparent Km values of 0.06 mm and 0.05 mm with enzymes I and II using tRNApC as cosubstrate. Both tRNA nucleotidyltransferases could utilize Mg++, Mn++, and Co++ to satisfy the requirement for divalent cation, but the latter two ions were less effective than Mg++. The rate of nucleotide incorporation into tRNAs with defined termini, as well as into other nucleic acids, was examined. tRNApCpCpA, rRNA, calf thymus DNA, or poly(C) were inactive as acceptors for either AMP, CMP, or UMP. tRNApCpC was a substrate for AMP incorporation, but was inactive with CTP or UTP. tRNApC and tRNApX were substrates for both CMP and UMP incorporation, but displayed diminishing activity in the presence of ATP. The apparent Km values for the various tRNA substrates were in the range of 4 to 30 µm, and were increased by the incorrect triphosphate. Liver tRNA nucleotidyltransferase showed no species specificity for the source of tRNA, being equally active with material from rabbit liver, yeast, and Escherichia coli. The extents of AMP, CMP, and UMP incorporation into tRNApC catalyzed by each enzyme were essentially the same suggesting that both enzymes probably acted on the same tRNA population. Another difference between the two tRNA nucleotidyltransferases was their response to a variety of salts. AMP incorporation catalyzed by either enzyme was inhibited by all salts examined. On the other hand, CMP incorporation catalyzed by Peak I was relatively little affected by monovalent salts at ionic strengths below 0.25, and that catalyzed by Peak II was greatly stimulated. These results point out that although many of the properties of the two enzymes are quite similar, significant differences do exist. A possible model for tRNA nucleotidyltransferase action is also presented.