PMID:11551215
| Citation |
Boehlein, SK, Nakatsu, T, Hiratake, J, Thirumoorthy, R, Stewart, JD, Richards, NG and Schuster, SM (2001) Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B. Biochemistry 40:11168-75 |
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| 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. |
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| Keywords |
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 |
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