Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2), performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase present in two different formulations, and its relationship with Laspartic acid, which is present in Aginasa, but not in Leuginase. The L-Asp present in Aginasa formulation was found to provide to EcA2 a resistance to in vitro proteolysis. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. Ion-mobility spectrometry?mass spectrometry reveals two conformers for the monomeric EcA2, and that monomeric species has sufficient capacity for selective binding to L-Asp and L-Glu. The N-terminal loop of the EcA2 present in Leuginase, which is part of the active site is disordered, but it gets ordered in the presence of L-Asp, while L-Glu only does so to a limited extent. These data provide new insights on the mechanistic of ligand recognition by EcA2, and the impact of formulation in its conformational diversity landscape.

Automated summary

The hydrolysis of asparagine and glutamine by L-asparaginase has been used in treating acute lymphoblastic leukemia for over four decades. A comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2) showed that the presence of L-Asp in the Aginasa formulation provides resistance to in vitro proteolysis for EcA2. Additionally, the study revealed the oligomeric landscape and conformational diversity of EcA2, as well as insights into ligand recognition by the enzyme.