Publications

• Larsen T.M., Boehlein S.K., Schuster S.M., Richards N.G.J., Thoden J.B., Holden H.M., Rayment I.

  Biochemistry 39:7330-7330(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Channeling of substrates and intermediates in enzyme-catalyzed reactions.

  Huang X., Holden H.M., Raushel F.M.

  The three-dimensional structures of tryptophan synthase, carbamoyl phosphate synthetase, glutamine phosphoribosylpyrophosphate amidotransferase, and asparagine synthetase have revealed the relative locations of multiple active sites within these proteins. In all of these polyfunctional enzymes, a ... >> More

  The three-dimensional structures of tryptophan synthase, carbamoyl phosphate synthetase, glutamine phosphoribosylpyrophosphate amidotransferase, and asparagine synthetase have revealed the relative locations of multiple active sites within these proteins. In all of these polyfunctional enzymes, a product formed from the catalytic reaction at one active site is a substrate for an enzymatic reaction at a distal active site. Reaction intermediates are translocated from one active site to the next through the participation of an intermolecular tunnel. The tunnel in tryptophan synthase is approximately 25 A in length, whereas the tunnel in carbamoyl phosphate synthetase is nearly 100 A long. Kinetic studies have demonstrated that the individual reactions are coordinated through allosteric coupling of one active site with another. The participation of these molecular tunnels is thought to protect reactive intermediates from coming in contact with the external medium. << Less

  Annu Rev Biochem 70:149-180(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.

  Tesson A.R., Soper T.S., Ciustea M., Richards N.G.

  Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagin ... >> More

  Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases. << Less

  Arch. Biochem. Biophys. 413:23-31(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.

  Larsen T.M., Boehlein S.K., Schuster S.M., Richards N.G.J., Thoden J.B., Holden H.M., Rayment I.

  Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant ... >> More

  Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase. << Less

  Biochemistry 38:16146-16157(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanistic issues in asparagine synthetase catalysis.

  Richards N.G., Schuster S.M.

  The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carbo ... >> More

  The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred. << Less

  Adv Enzymol Relat Areas Mol Biol 72:145-198(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.

  Boehlein S.K., Richards N.G., Schuster S.M.

  The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS- ... >> More

  The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants. In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue. Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis. As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R. D., Richards, N. G. J., and Schuster, S. M. (1993) J. Biol. Chem. 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B. In the case of C1A, the inhibition pattern suggests that an abortive complex is formed. This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS) << Less

  J. Biol. Chem. 269:7450-7457(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Asparagine biosynthesis by the Novikoff Hepatoma isolation, purification, property, and mechanism studies of the enzyme system.

  Patterson M.K. Jr., Orr G.R.

  J Biol Chem 243:376-380(1968) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.