Mapping the Aspartic Acid Binding Site of Escherichia coli Asparagine Synthetase B Using Substrate Analogs

Abstract

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of the aspartate binding site of Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction conditions for the stereospecific beta-functionalization of aspartic acid diesters, have been found to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog, demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal that large steric alterations in the substrate are not tolerated by the enzyme, consistent with the failure of previous efforts to develop AS inhibitors using random screening approaches, and that all of the ionizable groups are placed in close proximity in the bound conformation of aspartate.

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

“…To verify that these increases in the Michaelis constant reflected, at least in part, a decreased ability of the enzyme to bind aspartate, we examined whether CSA binding to each AS-B mutant was similarly reduced. In previous studies, we have shown that CSA is a competitive inhibitor with respect to only aspartate and no other AS-B substrate (26). With the one notable exception of the D320A AS-B mutant, we observed a good correlation between increases in K m (app) for aspartate and the loss of the ability of CSA to inhibit asparagine synthesis (Table VI).…”

“…This behavior supports the idea that mutations raising K m (app) for aspartate reflect an involvement in aspartate recognition and binding by the cognate residue in the wild-type enzyme. In experiments employing conformationally constrained amino acids, we have demonstrated that in the bound conformation of aspartate, all of the ionizable groups are located on one face of the substrate (26). It is therefore possible that the side chains of Thr-318 and Val-321 are positioned to interact with the hydrophobic face of the bound aspartate.…”

“…To investigate whether increases in aspartate K m were due primarily to impaired aspartate binding, we compared the ability of cysteinesulfinic acid (CSA) to inhibit the wild-type and mutant enzymes. CSA, an aspartate analog (25), is a competitive inhibitor with respect to aspartic acid in AS-B-catalyzed asparagine synthesis, having a K I value of 1.45 mM, but is not competitive with respect to any of the other substrates (26). Since it was unlikely that the substrate binding order for the AS-B mutants differed from wild-type AS-B, we obtained relative K I values for CSA by plotting the reciprocal of initial velocity versus CSA concentration, ensuring that substrate concentrations were maintained at a constant ratio (2-fold) to the appropriate K m (app) , making the resulting K I values directly comparable.…”

Asparagine Synthetase Chemotherapy

“…In this model, PP i was positioned over the pyrophosphatase loop region of the enzyme so that (a) the hydrogen bonding interactions with the enzyme corresponded to those observed in the GMPS/AMP/PP i crystal structure (48), and (b) noncovalent interactions with the active site Mg 2+ ion were maintained. Further MD refinement and energy minimization then gave a computational model for the AS-B/βAspAMP/PP i complex ( Figure 5), which was checked for consistency with the results of mutagenesis and kinetic studies employing AS-B (19,34,131,(134)(135)(136). Importantly for inhibitor development, we were able to identify completely conserved residues having side chains positioned within 5 Å of either βAspAMP or PP i .…”

“…First, by analogy to the chemistry of aminoacyl tRNA synthetases, attack of the side-chain carboxylate of bound aspartate on the α-phosphate of ATP likely proceeds via in-line attack with inversion at the phosphorus atom (Scheme 2) (128). Second, studies with non-natural, conformationally constrained aspartate analogs, prepared using the diastereoselective alkylation of L-aspartate diester derivatives (129,130), demonstrated that the polar functional groups are located on one face in the bound conformation of aspartate (131). After refinement using constrained molecular dynamics (MD) simulations (132) in combination with simulated annealing algorithms (133), and subsequent energy minimization, the resulting model was then modified by connecting the side-chain carboxylate of aspartate to the α-phosphate of the ATP moiety to form the βAspAMP intermediate.…”