Glycyl-tRNA synthetase from baker's yeast. Interconversion between active and inactive forms of the enzyme.
Glycyl-tRNA synthetase from baker's yeast has been purified to homogeneity. This synthetase was found to be very sensitive to proteases present in the yeast extracts and to oxidizing agents of thiol groups. In the absence of protease inhibitors and/or dithioerythritol, the enzyme rapidly lost its activity and could not be isolated. The use of these protectors allowed us to obtain different oligomeric structures of the synthetase. In the presence of a minimal concentration of dithioerythritol but in the absence of protease inhibitors, a tetrameric glycyl-tRNA synthetase of the alpha 2 beta 2 type (alpha = 67 600, beta = 57 500) with a very low specific activity was recovered. With high concentrations of both protectors, a dimeric enzyme was isolated with a specific activity comparable to that for other yeast synthetases. The enzyme was of the alpha 2 type where alpha = 70 000--80 000 daltons, depending on whether phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate was used as the protecting agent. The native form of the enzyme (alpha 2 = 160 000) associated easily with other proteins in various complexes of molecular weights from 250 000 to 300 000, some of them containing valyl-tRNA synthetase. The dimeric glycyl-tRNA synthetase was found in equilibrium with its subunits. Diluting the enzyme solution or increasing the salt concentration displaced the equilibrium toward the monomers, which are catalytically inactive for both the tRNA aminoacylation and the PPi-ATP exchange reactions. Addition of both tRNAGly and ATP.MgCl2 plus glycine displaced the equilibrium toward the dimeric form of the enzyme. Thiol groups were found to be involved in the association between the two subunits and in both activities of the synthetase. The results are interpreted in the light of possible regulatory mechanisms of the activity of this synthetase.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006032:Glycine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D009994:Osmolar Concentration; D010710:Phosphates; D011189:Potassium Chloride / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012492:Salts
D010710:Phosphates; D012492:Salts; D011189:Potassium Chloride; D004228:Dithionitrobenzoic Acid; D000604:Amino Acyl-tRNA Synthetases; D006032:Glycine-tRNA Ligase