Limited proteolysis of the tryptophanyl-tRNA synthetase

Abstract Earlier studies have shown that native tryptophanyl-tRNA synthetase from beef pancreas is composed of two apparently identical subunits having a molecular weight of 60 000 ± 2000 each. Incubation of the purified enzyme with trypsin under restrictive conditions results in splitting of each subunit to form an enzymatically inactive polypeptide chain of mol. wt 24 500 ± 1500 . During proteolysis, two distinct intermediate forms of mol. wt 51 000 ± 2000 and 40 000 ± 2000 and fragments of mol. wt 14 000 ± 2500 are formed. The presence of substrates, viz. ATP, tryptophan or tryptophanyl adenylate, decreases the rate of proteolysis. However, a band pattern monitored by acrylamide gel electrophoresis is qualitatively indistinguishable from that obtained in the absence of substrates. Native and trypsin-modified subunits (the latter having a molecular weight of 24 500) have been maleylated, reduced, carboxymethylated and subjected to exhaustive digestion by trypsin followed by peptide mapping. Comparison of the finger prints has shown that the trypsin-modified subunit represents a polypeptide with lowered content of dicarboxylic amino acids. That the number of peptides revealed after complete proteolysis of native and trypsin-modified subunits does not favour the presence of long repetitive sequences in each subunit, is at variance with some bacterial aminoacyl-tRNA synthetases. Study of the fluorescence polarisation of 1-anilino-8-naphthalene sulphonate adsorbed on the dimeric tryptophanyl-tRNA synthetase, indicates that the molecule behaves as a complete entity in Brownian rotation. The trypsin-resistant end products, composed of two types of polypeptides (mol. wts 24 500 and 14 000), remain associated with each other. From the mol. wt of this associate it follows that each fragment is present in the associate in duplicate. When the purification procedure was carried out in the absence of a protease inhibitor, the active modified enzyme form was obtained. As judged from the molecular weight values, it is composed of two equal subunits corresponding to one of the products of limited proteolysis. The data presented are compatible with compact three-dimensional structure of tryptophanyl-tRNA synthetase having very limited regions exposed to exogenous or endogenous proteolysis.