Purification and characterization of histidyl-transfer RNA synthetase from Neurospora crassa.
Histidyl-tRNA synthetase (L-histidine:tRNAHis ligase (AMP-forming), EC 6.1.1.21) has been purified 921-fold from crude extracts of lyophilized mycelia of Neurospora crassa. Sodium dodecyl sulfate gel electrophoresis at pH 8.9 of the purified enzyme yields one band with an apparent Mr of 62 500. The estimated Mr by Sephadex gel filtration is 125 000. Thus the native histidyl-tRNA synthetase of N. crassa is a dimer, composed of two identical subunits. The Km values determined in the enzyme-catalyzed esterification of [14C]-histidine to tRNAHis are: for histidine, 5.8 x 10(-6 M, for ATP, 5.9 x 10(-4) M, and for tRNAHis, 1.2 x 10(-7) M. Effects of various intermediates of the histidine, tryptophan and arginine biosynthetic pathways on histidyl-tRNA synthetase activity were studied. The Ki values for imidazoleglycerol phosphate and histidinol (histidine intermediates and competitive inhibitors of the enzyme) are 1.1 x 10(-2) M, 1.3 x 10(-6) M, respectively. The Ki for indoleglycerol phosphate (a tryptophan intermediate and non-competitive inhibitor) is 1.2 x 10(-3) M.
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D006643:Histidine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010940:Plant Proteins / Q000302:isolation & purification*
D010940:Plant Proteins; D000604:Amino Acyl-tRNA Synthetases; D006643:Histidine-tRNA Ligase