Purification and characterization of histidyl-transfer RNA synthetase from Neurospora crassa

Abstract

Histidyl-tRNA synthetase ( l-histidine:tRNA His 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 M r of 62 500. The estimated M r 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 K m values determined in the enzyme-catalyzed esterification of [ 14C]-histidine to tRNA His are: for histidine, 5.8·10 −6 M , for ATP, 5.9·10 −4 M , and for tRNA His, 1.2·10 −7 M. Effects of various intermediates of the histidine, tryptophan and arginine biosynthetic pathways on histidyl-tRNA synthetase activity were studied. The K i values for imidazoleglycerol phosphate and histidinol (histidine intermediates and competitive inhibitors of the enzyme) are 1.1·10 −2 M, 1.3·10 −6 M, respectively. The K i for indoleglycerol phosphate (a tryptophan intermediate and non-competitive inhibitor) is 1.2·10 −3 M.