Phenylalanyl-tRNA synthetase from baker's yeast. Salt dependence of steady-state kinetics indicates two molecular forms of the enzyme.
Steady-state kinetic data of aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase depend on salt concentration. At 5 mM KCl and 20 mM MgSO4 a non-linear curve is found in the double-reciprocal plot for ATP and phenylalanine, while at 200 mM KCl and 50 mM MgSO4 a linear curve is observed. KCl and MgSO4 dependence of the reaction also show biphasic curves with intersection points of the two extrapolated linear parts at 50 mM and 10 mM, respectively. A biphasic curve is also found if the concentration of CTP is varied at constant low ATP concentration. Extrapolations of the linear parts of the curves for ATP as well as for CTP at 5 mM KCl and 20 mM MgSO4 intersected the 1/[NTP] axis at 1.2 +/- 0.1 mM. Hence the existence of a non-linear curve for ATP as well as phenylalanine does not necessarily indicate two non-equivalent binding sites for these substrates. A more likely explanation is the existence of two different molecular forms of phenylalanyl-tRNA synthetase which are interconvertible by salt. This explanation is substantiated by the observation that proteolytic digestion of phenylalanyl-tRNA synthetase is more easily achieved at low than at medium ionic strength. In addition mischarging of tRNAIle with phenylalanine by phenylalanyl-tRNA synthetase occurs at a moderate rate at 5 mM KCl and 20 mM MgSO4 whereas it is largely depressed by addition of either 5 mM CTP or 150 mM KCl.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D007527:Isoenzymes; D011189:Potassium Chloride; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium