Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of subsites for donor nucleotide triphosphates.
tRNA nucleotidyltransferase incorporates both AMP and CMP into tRNA acceptors. Studies of the effects of nucleoside triphosphates, nucleotide analogues, and affinity reagents on AMP and CMP incorporation indicate that these residues are donated from different subsites. However, neither of these sites is completely specific for nucleoside triphosphate binding, and CMP can actually be incorporated from the AMP-donating site, although at a slow rate. The two donor subsites interact with each other, such that binding of a ligand to the ATP site stimulates incorporation from the CMP-donating site. This interaction accounts for the biphasic CTP saturation curve and the unusual effects of nucleoside triphosphates on CMP incorporation observed earlier. In addition to donating CMP, the CTP subsite also serves as the position of binding of the terminal C residue of tRNA-C-C and, in the absence of CTP, for binding of the terminal residue of tRNA-C. These results, together with those in the accompanying paper, have defined multiple accepting and donating subsites within the active site of tRNA nucleotidyltransferase, as predicted from our previous model for enzyme action (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468). However, since we have been unable to obtain definitive evidence for two CMP-donating sites, we have considered a modification of this earlier model which utilizes only a single CMP-donating site. Using these models, we discuss how the specificity of the donor and acceptor subsites ensures the accurate synthesis of the -C-C-A sequence of tRNA.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003568:Cytidine Monophosphate / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011817:Rabbits; D012265:Ribonucleotides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012265:Ribonucleotides; D000249:Adenosine Monophosphate; D003570:Cytidine Triphosphate; D012316:RNA Nucleotidyltransferases; D003568:Cytidine Monophosphate