The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship.
Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope-exchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the aminoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gln and of the the deacylation of glutaminyl-tRNA Gln. At acidic pH, glutaminyl-tRNA synthetase catalyzes the synthesis of the glutaminyl-tRNA Gln and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating than glutaminyl-tRNA Gln cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.
D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005975:Glutamate-tRNA Ligase / Q000378:metabolism; D005973:Glutamine / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D013095:Spermidine / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D005973:Glutamine; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase; C020659:glutaminyl-tRNA synthetase; D013095:Spermidine