Purification and Properties of the Asparagine Synthetase of Streptococcus bovis

Streptococcus bovis is an unusual member of the genus Streptococcus because it can synthesize its major cellular nitrogen compounds from ammonium salts (1).Other members of the genus are well known for their requirements for amino acids.Bn enzyme survey was conducted in order to provide information concerning the primary sites of incorporation of NHa into organic nitrogen in Streptococcus bovis.During the course of this survey, it was found that cell extracts were capable of catalyzing the formation of a hydroxamate of aspartic acid in the presence of adenosine triphosphate and Mg++, whereas no similar reaction could be detected with glutamic acid as substrate.These observations suggested the possibility that the extracts contained an asparagine-synthesizing system and were devoid of glutamine synthet.ase.The aspartate-activating system was purified and shown to be an asparagine synthetase which catalyzes the following reaction.ATP + L-aspartate + NH3 + L-asparagine + AMP + PPi (1) Ravel, Norton, Humphreys, and Shive (2) have demonstrated the presence of an asparagine synthetase in Lactobacillus arabinosus which catalyzes the above reaction.The S. bovis enzyme appears to be very similar to the L. arabinosus enzyme.The present report is concerned with the purification and properties of the asparagine synthetase of S. bovis.EXPERIMENTAL PROCEDURE ,1PateriaZs-Crystalline adenylate kinase, pyruvate kinase, and lactic dehydrogenase were obtained from California Corporation for Biochemical Research.Inorganic pyrophosphatase was prepared according to the alternative purification procedure described by Heppel (3) through the ethanol fractionation step.Crystalline inorganic pyrophosphatase was obtained from Worthington Biochemical Corporation.P-Aspartylhydroxamic acid was prepared according to the met#hod of DeGroot and Lichtenstein (4).DL-Isoasparagine was kindly provided by Dr. F. H. Carpenter.DEAE-Sephadex and Sephadex G-50 were obtained from Pharmacia Corporation and DEAE-cellulose was obtained from Carl Schleicher and Schuell Corporation.Chemical and Enzymatic Determinations-Aspartylhydroxamic acid was determined with FeC13 (5).ADP was measured by a phosphoenolpyruvate, pyruvate kinase, NADH, and lactic dehydrogenase coupled system (6) with 1 unit of each crystalline enzyme in a l-ml reaction mixture containing 33 mM Tris buffer, pH 7.1, 1.7 mM phosphoenolpyruvate, and 0.33 mM NADH.AMP was determined in an ident.icalassay system with excess