A kinetic and structural characterization of adenosine-5'-triphosphate: ribonucleic acid adenylyltransferase from Pseudomonas putida.
A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made. During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed. Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties. Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies. Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S. The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000. Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification. The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml). The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases. The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M. Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo [2,1-i] purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B. Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction. The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA. The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology*; D012294:Rifamycins / Q000494:pharmacology
D012294:Rifamycins; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase