Purification and characterization of beef pancreatic asparagine synthetase

Journal of Biological Chemistry

et al. 1997

Self Cite

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of ␤-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wildtype AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation ␤-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to ␤-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

mentioning

confidence: 51%

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Boehlein

Walworth

Journal of Biological Chemistry

et al. 1997

Self Cite

“…The detailed kinetic and structural characterization of ASNS from mammalian sources historically proved to be difficult because of both reported low abundance and instability of the native enzyme during purification (25)(26)(27)(28)(29). In addition, only small amounts of recombinant, wild-type human ASNS could be obtained in a variety of early expression systems, the enzyme being purified to homogeneity using an affinity chromatography protocol (30)(31)(32).…”

Section: Structure Of Asparagine Synthetasementioning

confidence: 99%

“…The complex three-dimensional fold of the C-terminal domain of AS-B is similar to that observed in ATP pyrophosphatases (47), an enzyme superfamily that includes guanosine-5′-monophosphate synthetase (GMPS) (48), argininosuccinate synthetase (49), ATP sulfurylase (50), carbapenam synthetase (51), β-lactam synthetase (BLS) (52,53), and ThiI (54). Because all ATP pyrophosphatases convert ATP to AMP, the C-terminal active site of ASNS likely catalyzes activation of the side-chain carboxylate of aspartate to form an electrophilic intermediate, β-aspartyl-AMP (βAspAMP) 1, and inorganic pyrophosphate (PP i ) (Scheme 2) (28,55). As observed in other glutaminedependent amidotransferases (44,46,(56)(57)(58)(59)(60)(61), the two active sites of AS-B are linked by a solvent-inaccessible, intramolecular "tunnel" that is sufficiently wide to allow passage of an ammonia molecule (Figure 1b) (62).…”

Section: Structure Of Asparagine Synthetasementioning

confidence: 99%

Asparagine Synthetase Chemotherapy

Kilberg

2006

Annu. Rev. Biochem.

204

221

Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzymebased methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

“…Amtno aetds are denoted using the standard one-letter code AMP intermediate [15]. Building upon our purification and charactertzation of beef pancreaUc asparagme synthetase [21], we have sequenced the gene encoding asparagine synthetase in E. coli ( Fig. I) [22].…”

Section: Introductionmentioning

confidence: 99%

An alternative mechanism for the nitrogen transfer reaction in asparagine synthetase

Schuster

1992

FEBS Letters

Self Cite

In the absence of crystallographic data, :he roechambm of mtrogen transfer from glatalnin¢ in ahparagine synthetase (AS) remain~ under active investigation. Surprisingly, tl~¢ glutamme-dependent AS from E~chet tchta colt (AsnB) appears to lack a conserved histidme residue, necessary for mtrogea transfer if the reaction proceeds by the accepted pathway :n other glutamine amidotransferases, but retains the abdity to synthe.~lze asparasine. We propose an alternative n~echanisrn for nltrogen transfer m AsnB wh)ch obviates the reqmrement for participation of histtdme m this step. Our hypothesis may also be more generally applicable to other glutalaame-dependent amidotransferases.