Affinity Chromatography on Agarose-hexyl-adenosine-5'-phosphate of Methionyl-tRNA Synthetase from Escherichia coli. Application of the Couplings between the Methionine and ATP Sites

Abstract

Recent studies by us [Biochemistry (1977) 16, 2570-25791 have shown that L-methioninol, a methionine analog lacking the carboxylate negative charge, enhances the affinity of AMP for methionyl-tRNA synthetase while L-methionine antagonizes the nucleotide binding.Such couplings between ligands of the enzyme have now been applied to affinity chromatography of methionyl-tRNA synthetase on an agarose-hex~l-adenosine-5'-phosphate gel (the spacer is attached to AMP at the adenine C-8 position). Retention of the enzyme on this gel column was shown to be dependent on the presence of appropriate concentrations of magnesium and of L-methioninol in the equilibration buffer. The enzyme was then specifically recovered from the column by omitting the amino alcohol or by adding an excess of L-methionine which antagonizes the cooperative effect of L-methioninol.This approach has provided the basis for a new purification procedure of methionyl-tRNA synthetase which leads to a 200-fold purification in a single chromatographic step. In this manner, after 30 -50% ammonium sulfate fractionation of extracts of Escherichiu coli EM 20031 (carrying the F32 episome), 0.25mgxmethionyl-tRNA synthetase was obtained at 90% purity per ml of agarose-hexyI-adenosine-5'-phosphate gel.Several affinity chromatographic methods have already been reported in the case of aminoacyl-tRNA synthetases. These methods are based on the immobilization of either the specific tRNA [l -51, amino acid [6] or aminoalkyl-adenylate [7]. Recently a method for the purification of isoleucyl-tRNA synthetase on blue dextran-Sepharose has been described [8] which is assessed to be affinity chromatography [9].A new approach is now reported which is based upon the couplings (i.e. the change in the dissociation constant of one ligand upon ligation of another ligand) between ligands within the adenylating sites of Escherichiu coli activating enzymes [lo-141. In the case of methionyl-tRNA synthetase, the ligand may couple either synergistically or antagonistically within the enzymic site [14]. For example, L-methioninol, an L-methionine analog lacking the carboxylate function, enhances the binding of AMP while L-methionine itself antagonizes this binding. An abstract of this work was presented at the 10th InternationalEnzyme. Methionyl-tRNA synthetase or L-methionine: tRNAMe' Congress of Biochemistry (Abstract 03-2-1 13).ligase (AMP forming) (EC 6.1.1.10).Such couplings have now been demonstrated to apply to affinity chromatography of methionyl-tRNA synthetase. The AMP nucleotide is immobilized on agarose via the C-8 of the adenine and a six-carbon spacer (hexane) [15]. Substitution of the nucleotide at this position is known not to obliterate binding to the enzyme [12,16]. Methionyl-tRNA synthetase may be specifically bound to the immobilized AMP molecule in the presence of L-methioninol and magnesium in the equilibration buffer. Selective recovery of the enzyme is then achieved by addition of L-methionine. MATERIALS AND METHODSHomogeneous native and trypsin-modified methio...

Methionyl‐tRNA Synthetase from Escherichia coli

“…nucleoside complexes indicating that the H-2 proton is probably close to protons of the protein by which it might be relaxed. This fits well with the fact that analogs of adenosine modified at C-2 of the purine ring bind little, if at all, to the enzyme [12], whereas analogs modified at C-8 (even with bulky groups) still bind well [12,13].…”

Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

“…synthetase (Fayat et al, 1977), kindly supplied by Dr. Mildred Cohn, was used in these experiments. Optimal conditions for the rate studies with each of the three tRNAs, normal methionine tRNA and the two forms of FUra-substituted tRNAf161, were determined by examining the magnesium ion requirement of the reaction at various KC1 concentrations.…”

“…The kinetics of methionyl-tRNA formation were determined in a reaction mixture containing 0.04 M Hepes, pH 7.5, 0.15 M KC1, 0.002 M ATP, 0.001 M dithiothreitol, 0.007 M magnesium acetate, 1 X 10~4 M EDTA, 0.1 mg/mL bovine serum albumin, 8 X 10'5 M [14C]methionine (51.4 mCi/ mmol), 180 pmol of purified methionyl-tRNA synthetase (Fayat et al, 1977), kindly supplied by Dr. Mildred Cohn, and varying amounts of tRNA. Reaction mixtures were preincubated at 25 °C for 1 min before addition of the enzyme.…”

Aminoacyl-tRNA Synthetases: General Features and Recognition of Transfer RNAs

“…Undoubtedly these studies will continue to refine our understanding of the mechanistic aspects of the synthetases and define better the differences and similarities in the mechanism of various enzymes. Efforts are also being directed at improving the purifi cation methods of the synthetases (330)(331)(332). Taking advantage of agarose-hexyl-adenosine-5'-phosphate and blue dextran-sepharose (330,331), affinity chromatogra phy has been used with good success.…”

“…Efforts are also being directed at improving the purifi cation methods of the synthetases (330)(331)(332). Taking advantage of agarose-hexyl-adenosine-5'-phosphate and blue dextran-sepharose (330,331), affinity chromatogra phy has been used with good success. Some of these promising procedures will certainly be adopted by many workers and further improved.…”