Potent Transition-State Analogue Inhibitor of Esherichia coli Asparagine Synthetase A (MOLECULAR BIOFUNCTION-Functional Molecular Conversion)

Abstract

A potent and slow-binding inhibitor of E. coli asparagine synthetase A (AS-A) was synthesized, and its inhibition behavior was characterized. The enzyme complexed with the inhibitor was analyzed by X-ray diffraction analysis to identify several key amino acid residues responsible for catalysis as well as for substrate recognition. AS-A catalyzes the formation of L-Asn from L-Asp and ammonia coupled with the hydrolysis of ATP to AMP and pyrophosphate. The reaction catalyzed by this enzyme is prototypic of mammalian asparagine synthetase (AS-B) which utilizes glutamine as a nitrogen source. In addition, asparagine synthetase is a potential target for chemotherapy to treat certain leukemias. We therefore designed and synthesized a transition-state analogue, N-adenylated S-methyl-L-cysteine sulfoximine 1, based on the proposed reaction mechanisms of AS-A. The compound 1 strongly inhibited the E. coli AS-A in a time-dependent manner with an overall inhibition constant (Ki*) of 67 nM and with an onset rate of inactivation of 3.27 s-1 mM-1. The inhibition was almost irreversible and no regain of enzyme activity was observed in 10 days after gel filtration. The inhibitor 1 was also used as a ligand for X-ray diffraction analysis of AS-A. The X-ray crystal structure of AS-A complexed with 1 revealed several key amino acid residues such as Arg 100, Gln 116 and Asp 46 responsible for catalysis as well as those for substrate recognition. An attempt to inhibit AS-A by each diastereomer of S-methyl-L-cysteine sulfoximine and ATP is also described. Since AS-A is prototypic of asparagine synthetases in terms of the chemistry in substrate activation, compound 1 should formulate a basis for future inhibitor design of asparagine synthetase B.