title: Purification and properties of phenylalanyl-tRNA synthetase from a higher plant (Phaseolus vulgaris).
abstract: Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps. The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da). Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance. Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65. The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group. tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated. No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E. coli, yeast and hen liver. ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety.
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D007887:Fabaceae / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010652:Phenylalanine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase