Purification and Properties of Phenylalanyl-tRNA Synthetase from Baker's Yeast

In an effort to avoid proteolytic fragmentation of enzymes extracted from yeast cells, the (L-phenylalanine:tRNAPhe ligase (AMP-forming) phenylalanyl-tRNA synthetase (EC 6.1.1.20)) has been isolated from toluene lysates of baker's yeast in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride. The procedure includes ammonium sulfate fractionation and chromatography on DEAE-cellulose and hydroxyl-apatite columns. Acrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate indicates a single subunit of 75 000; other isolations have led to two subunits of 75 000 and 63 000, respectively, in agreement with other workers. Steady state kinetic analysis of the enzyme has also been carried out. The apparent kinetic patterns resulting from application of Cleland's procedure, in which the substrates are varied pair-wise in the presence of saturating concentrations of the third component, suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion but do not completely eliminate a random mechanism.