Inhibition, characterization, and crystallization of glutamine-dependent asparagine synthetase

Acute lymphoblastic leukemia is commonly treated using the enzyme L-asparaginase, which hydrolyzes asparagine to aspartic acid, leaving the tumor cells unable to obtain asparagine from the circulating plasma. Several lines of evidence suggest that resistance to L-asparaginase treatment is correlated to up-regulation of the enzyme responsible for the biosynthesis of asparagine, asparagine synthetase. Recent work has shown that a sulfoximine derived inhibitor of asparagine synthetase with nanomolar potency can suppress the proliferation of drug-resistant MOLT-4 leukemia cells in the presence of L-asparaginase, a result that supports the use of inhibitors of asparagine synthetase in the clinical treatment of acute lymphoblastic leukemia. This work strives to develop second-generation inhibitors of asparagine synthetase by gaining a better understanding of the active site-inhibitor interactions, reaction mechanism, and structure of this enzyme.;Sulfoximine derived inhibitors of asparagine synthetase were designed to increase the bioavailability and potency of the molecule. Steady-state kinetic analysis of these compounds found that a localized negative charge on the inhibitor that mimics the phosphate group is essential to ligand binding in asparagine synthetase. These findings place an important constraint on the design of future inhibitors of asparagine synthetase.;Work to understand the reaction mechanism and validate a computational model of aspirating synthetase focused on a conserved glutamate residue which was hypothesized to act as the general base for the deprotonation of ammonia in the transition state. However, site-directed mutagenesis, kinetic analysis, and isotopic labeling studies of the glutamine-dependent asparagine synthetase from E. coli found that this residue is critical to formation of the betaAspAMP intermediate.;Finally in an effort to understand the structure-function relationships of asparagine synthetase, we sought to obtain a high resolution crystal structure of asparagine synthetase. Through the preparation of a doubly inhibited form of the enzyme, we hoped to lock the enzyme into an active conformation that promoted crystallization. Unfortunately, preliminary crystallization trials did not produce crystals suitable for diffraction but important information regarding the stability of the enzyme was obtained.