Intramolecular tunnel and regulatory mechanisms of asparagine synthetase (ASNS).

摘要

So far, more and more evidence from several actively studied polyfunctional enzymes indicate the existence of an intramolecular tunnel. Among those enzymes, ammonia/ammonium is the most common intermediate thus far. As one of these enzymes, the tunnel in asparagine synthetase B (ASB) from Escherichia coli is about 20 A long. It connects two active sites, a glutaminase site and a synthetase site.; My work on asparagine synthetase (ASNS), an enzyme involved in leukemia resistance to the treatment using asparaginase, has focused on the following aspects: (1) kinetic characterization of ASNS; (2) structure activity relationships; (3) regulatory mechanisms.; In this work, I developed a first quantitative NMR based assay (K. Li et al., Biochemistry, 2007, 46 [16], 4840-4849) and studied the efficiency of ammonia tunneling in ASB catalyzed reaction.; The function of bioactive macromolecule can only be understood under the structural context. Therefore, to study the function of the intramolecular tunnel in ASB, first I identified the tunnel residues by computational methods. Most of the tunnel residues are conserved from prokaryote to eukaryote species. The studies of several tunnel mutants showed that one of the mutants may have a blocked tunnel.; Among the family of amidotransferases, the two half reactions are strictly coupled. While ASNS can catalyze the hydrolysis of glutamine without other substrates, this raises the interest in the regulatory mechanisms of this enzyme, which prevents it from consuming glutamine and releasing free ammonia. As part of my PhD research, I investigated the mechanisms of product inhibition to this enzyme. Under physiological conditions, the activity of this enzyme may be inhibited significantly by asparagine, which prevents cells from wasting glutamine and producing toxic ammonia in vivo.; So far, only one crystal structure of glutamine dependent asparagine synthetase was reported. To further understand the function of ASNS, especially of human enzyme, more crystal structures are needed. Therefore, co-working with Alexandria Berry, I investigated the binding of substrate/transition state analog to hASNS and successfully prepared inhibitor bound enzyme. The prepared enzyme has been sent for crystal analysis.