Glycyl-tRNA synthetase from baker's yeast. Interconversion between active and inactive forms of the enzyme

Abstract

Glycyl-tRNA synthetase from baker''s yeast [S. cerevisiae] was purified to homogeneity. This synthetase was 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 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 found 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 MW 250,000-300,000, 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.cntdot.MgCl2 plus glycine displaced the equilibrium toward the dimeric form of the enzyme. Thiol groups were involved in the association between the 2 subunits and in both activities of the synthetase. The results are interpreted in light of possible regulatory mechanisms of the activity of this synthetase.