Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs.
The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity
D011756:Diphosphates; D013096:Spermine; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D008274:Magnesium