Strain-specific physicochemical behavior of asparaginase B from E coli: therapeutic implications

Asparaginases have been used for over four decades to treat acute lymphoblastic leukemia by hydrolyzing asparagine and glutamine. Escherichia coli L-asparaginase 2 (EcA2) monomers possess a capping loop that acts as a lid over the active site upon substrate binding. In this comparative study, we investigated two commercially available EcA2-containing drug formulations using a comprehensive set of biophysical and biochemical approaches. Our analysis focused on the oligomeric landscape, conformational dynamics, and plasticity of EcA2 in the presence of L-aspartic acid (L-Asp), which is present in Aginasa but absent in Leuginase. Of note, L-Asp in Aginasa confers resistance to in vitro proteolysis. EcA2 exhibited a composition of monomers and oligomers to tetramers that changed minimally in the presence of L-Asp. Ion mobility spectrometry-mass spectrometry revealed two conformers for monomeric EcA2, with the monomeric species exhibiting selective binding to L-Asp and L-glutamate (L-Glu). Moreover, the N-terminal loop of EcA2 in leuginase, an important part of the active site, showed disorder but was ordered in the presence of L-Asp, whereas L-Glu showed limited order. These results provide new insights into the mechanistic aspects of ligand recognition by EcA2 and highlight the influence of formulation on conformational diversity.