Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B

Biochemistry. 2001 Sep 18;40(37):11168-75. doi: 10.1021/bi0155551.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / pharmacology*
  • Adenosine Triphosphate / metabolism
  • Asparagine / biosynthesis*
  • Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / antagonists & inhibitors*
  • Cysteine / analogs & derivatives
  • Cysteine / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / enzymology*
  • Hydrolysis
  • Methionine Sulfoximine / analogs & derivatives
  • Methionine Sulfoximine / pharmacology*
  • Models, Chemical
  • Neurotransmitter Agents
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphorus Isotopes
  • Spectrometry, Mass, Electrospray Ionization


  • 5'-((methionine sulfoximinophospho)oxy)adenosine
  • Enzyme Inhibitors
  • Neurotransmitter Agents
  • Phosphorus Isotopes
  • Methionine Sulfoximine
  • Adenosine Monophosphate
  • Asparagine
  • Adenosine Triphosphate
  • Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor
  • asparagine synthetase (glutamine-hydrolyzing)
  • Cysteine
  • cysteine sulfinic acid