Anthranilate Synthetase from Serratia marcescens: ON THE PROPERTIES AND RELATIONSHIP TO THE ENZYME FROM SALMONELLA TYPHIMURIUM

Abstract

Abstract Anthranilate synthetase was purified from Serratia marcescens. The enzyme was homogeneous by the criterion of disc gel electrophoresis. Glutamine- and NH3-dependent anthranilate synthetase activities were subject to end product inhibition by tryptophan. Inhibition by tryptophan exhibited positive cooperativity. In the absence of tryptophan, Michaelis-Menten kinetics were obtained for saturation by the two substrates, chorismate and glutamine. Positive cooperativity for chorismate was detected in the presence of tryptophan. These kinetic properties of anthranilate synthetase from S. marcescens were similar to those of the multifunctional anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) aggregate from Salmonella typhimurium. A molecular weight of approximately 141,000 was estimated for the oligomeric anthranilate synthetase from S. marcescens. Nonidentical subunits of molecular weights approximately 60,000 and 21,000 were detected by disc gel electrophoresis in sodium dodecyl sulfate. The glutamine analogue, 6-diazo-5-oxo-l-norleucine (DON), and sulfhydryl reagents selectively inactivated glutamine-dependent anthranilate synthetase activity; NH3-dependent activity was largely retained. 14C-Labeled DON and iodo[14C]acetamide were incorporated into the small subunit thus establishing the relationship of this subunit to anthranilate synthetase Component II from anthranilate synthetase-PR transferase of S. typhimurium. Approximately 2 moles of DON or iodoacetamide were incorporated per mole of anthranilate synthetase, suggesting that the oligomeric enzyme contains two glutamine-binding subunits (anthranilate synthetase Component II). From a consideration of the approximate size of the native enzyme and its subunits a composition of two chains each of anthranilate synthetase Components I and II is suggested. A glutaminase activity was detected. This activity may serve to transfer the amide of glutamine from anthranilate synthetase Component II to the catalytic site on Component I. Catalytic properties and structural features of anthranilate synthetase from S. marcescens were compared to those of trypsin-treated anthranilate synthetase-PR transferase from S. typhimurium (Hwang, L. H., and Zalkin, H., J. Biol. Chem., 246, 2338 (1971)). On the basis of similarities between the two enzymes it is suggested that gene fusion could account for the two types of anthranilate synthetase.