[Tryptophanyl tRNA synthetase: isolation and characteristics of the tryptophanyl-enzyme].
The catalytic groups, involved in aminoacyl-tRNA formation remain unknown. The isolation and identification of an active covalent complex between the enzyme and substrate is an essential step in understanding the reaction mechanism. We identified and isolated the covalent complex of tryptophanyl-tRNA synthetase (EC 6.1.1.2) and tryptophane which was able to aminoacylate the tRNATrp in the absence of ATP. In beef pancreas tryptophanyl-tRNA synthetase preparations, isolated by the previously described method, a tightly bound tryptophan was revealed which could not be removed by charcoal treatment, by gel-filtration and by replacement with the excess of typtamine, a competitive inhibitor of tryptophane. This tightly bound tryptophane is able to exchange rapidly and specifically with radioactive tryptophane allowing to obtain [14C]tryptophane-tryptophanyl-tRNA synthetase complex. After the reaction of this complex with NH2OH at neutral pH tryptophanyl hydroxamate is formed proving the activated state of the tryptophane in the initial complex with the enzyme. No nucleotide impurites were noticed in the enzyme preparation; the complex is stable at denaturation. A conclusion is made that the tryptophanyl-tRNA synthetase isolated by our method is a tryptophanyl-enzyme. The tryptophanyl residue could be specifically transferred to tRNATrp in the absence of other substrates of the reaction, the efficiency of the transfer does not exceed 25%. The content of the covalently bound tryptophane never exceeds 1 mole per mole of the dimeric enzyme. The total content of tryptophane in the forms of tryptophanyl-enzyme and tryptophanyl adenylate enzyme complex equals 2 moles per mole of the enzyme. The tryptophanyl-enzyme is destroyed during incubation with AMP or with pyrophosphate. The role of the tryptophanyl-enzyme as a possible intermediate in the course of aminoacylation of tRNATrp is discussed.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D011756:Diphosphates; D006898:Hydroxylamines; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D014364:Tryptophan / Q000302:isolation & purification; D014369:Tryptophan-tRNA Ligase / Q000302:isolation & purification
D011756:Diphosphates; D006898:Hydroxylamines; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase
Triptofanil-tRNK-sintetaza: vydelenie i kharakteristika triptofanil-fermenta.