Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.
TRNA-Phe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNA-Phe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNA-Phe-C-C-2'dA and the tRNA-Phe-C-C-formycin-oxi-red, are strong competitive inhibitors for tRNA-Phe-C-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vmax to about 25%; but whereas tRNA-Phe-C-C-2dA has no influence on KmATP and Km Phe during ATP/PPi exchange, tRNA-Phe-C-C-formycin-oxi-red reduces KmATP from 1430 muM, found in the absence of tRNA-Phe, to 230 muM, and Km-Phe, from 38 to 14 muM. The values found in the presence of tRNA-Phe-C-C-formycin-oxi-red during ATP/PPi exchange are identical with those determined in the phenylalanylation of tRNA-Phe-C-C-A. All other tRNA-Phe species carrying a modified 3'end that cannot be phenylalanylated exhibit a mixed competitive-noncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence KmATP and KmPHE  and only weakly, if at all, Vmax. The results show that the 3'adenosine of tRNA-Phe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNA-Phe triggers a conformational change of the enzyme.
D000241:Adenosine / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D007700:Kinetics; D008433:Mathematics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D011485:Protein Binding; D012343:RNA, Transfer / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000032:analysis / Q000201:enzymology*; D013816:Thermodynamics
D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D000241:Adenosine