Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.
The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.
D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors*; D004396:Coloring Agents / Q000494:pharmacology*; D003911:Dextrans / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D007533:Isoleucine-tRNA Ligase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008433:Mathematics; D012343:RNA, Transfer
D004396:Coloring Agents; D003911:Dextrans; D012343:RNA, Transfer; C017080:blue dextran; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase