Nucleic Acids Research

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60—»-51—*40-»-24 kilodaltons. The transition 5V-MO is hindered in tryptophanyl adenylate*enzyme complex. Yeast tRNA^rP accelerates the first steps of hydrolysis and decelerates the transition 40-+-24. Once tRNA^rp is added to the synthetase*adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRUA^rp oxidized with NalOAand tRNAP lacking the 3'-terminal adenosine. Oxidized tRNAP (but not tRNAP without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNA^rp With beef pancreas tryptophanyl-tRNA synthetase involving the association of tRUA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate. INTRODUCTION Interaction of aminoacyl-tRNA synthetases with substrates (amino acids, ATP, tRNA) to yield aminoacyl-tRNAs is an intricate process consisting of many steps (see reviews ). To gain a better insight into these interactions, studies on the reaction kinetics as well as physico-chemical approaches (fluorescent titration, temperature jump, inhibition by substrate analogs) have been used. In this work, an attempt has been made to study changes in aminoacyl-tRNA synthetase upon its interaction with substrates using limited proteolysis of the protein with trypsin. This approach is possible due to the fact that beef pancreas trypto© Information Retrieval Limited 1 Falconberg Court London W1V5FG England 625 Nucleic Acids Research phanyl-tRNA synthetase (EC 6.1.1.2) is subjected to successive limited proteolysis yielding a number of discrete products. As has been found earlier , hydrolysis of each of the two identical subunits (mol.wt. of the native subunit is about 60,000 ' ) proceeds in succession from the N-terminus of the molecule according to the scheme 6O->51—*'40-»-(24+14) kilodaltons. On the basis of this scheme, we studied the effect of tryptophanyl adenylate and various yeast tRNAs on limited proteolysis of these enzyme«ligand complexes. MATERIALS AND METHODS Tryptophanyl-tRNA synthetase from beef pancreas was isolated in this laboratory as described elsewhere . Prior to use, the enzyme was subjected to acid precipitation to eliminate tryptophan covalently bound to the protein . The pH of the protein solution (10-15 mg/ml) was adjusted to 5»0 in the cold by adding 0.1 M Na-acetate buffer, and the solution was allowed to stand at 4°C for 30 min. The precipitate was collected by centrifugation, washed with 0.02 M Na-acetate buffer (pH 5.0) and H20, and dissolved in buffer containing 0.02 M tris-HCl (pH 7.5), 2 x 10 M EDTA and 2 x 10"^ 2-mercaptoethanol. Limited proteolysis with trypsin TPCK-treated (Merck) was conducted at 37°0 in 50 ill of buffer containing 0.01 M Na-phosphate buffer (pH 6.5) with 0.01 M MgClo and 0.001 M 2-mercaptoethanol (buffer A ) . The concentration of synthetase in a sample was 3 x 10" M. In all the samples with tRNA its concentration was 4 x 10"' M. The hydrolysis was stopped by adding an 10-̂ ul aliquot of the hydrolysate to 1 jil of 10% sodium dodecylsulfate and 1 ̂ ul of 2-mercaptoethanol, and the mixture was heated instantaneously in a boiling water bath for 2 min. A 8-jil aliquot of the solution was loaded on 5% polyacrylamide gel and electrophoresed in the preq sence of 0.1% SDS according to Weber and Osborn in a slab 0.6 mm thick at 80 V and 30 mA. The gels were stained with Coomassie brilliant blue R-250 (Serva) and washed with 1% CH^COOH. Unfractionated yeast tRNA lacking tRNAP (tRNA~P) w a s obtained by means of removal of tRNA P from the total preparation due to separation on the benzoylated DEAE-cellulose column ;