title: Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.
abstract: Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Boehlein
authors.forename: T
authors.initials: T
authors.lastname: Nakatsu
authors.forename: J
authors.initials: J
authors.lastname: Hiratake
authors.forename: R
authors.initials: R
authors.lastname: Thirumoorthy
authors.forename: J D
authors.initials: JD
authors.lastname: Stewart
authors.forename: N G
authors.initials: NG
authors.lastname: Richards
authors.forename: S M
authors.initials: SM
authors.lastname: Schuster
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001216:Asparagine / Q000096:biosynthesis*; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000037:antagonists & inhibitors*; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology*; D008956:Models, Chemical; D018377:Neurotransmitter Agents; D019906:Nuclear Magnetic Resonance, Biomolecular; D010759:Phosphorus Isotopes; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C436714:5'-((methionine sulfoximinophospho)oxy)adenosine; D004791:Enzyme Inhibitors; D018377:Neurotransmitter Agents; D010759:Phosphorus Isotopes; D008717:Methionine Sulfoximine; D000249:Adenosine Monophosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C019742:asparagine synthetase (glutamine-hydrolyzing); D003545:Cysteine; C013461:cysteine sulfinic acid