Biophysical characterization of two commercially available preparations of the drug containingEscherichia coliL-Asparaginase 2

Abstract

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 two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, 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 (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

Structural and biochemical properties of L‐asparaginase

“…Conclusions gained from these studies will be discussed later. Three recently deposited structures of EcAII were determined in order to assess differences between two formulations of the clinical drug (Aginasa vs. Leuginase), but they do not provide additional structural or functional insights [88]. Two recent medium-resolution and high-resolution structures (PDB IDs 6v5f and 6yzi, respectively) remain without accompanying publications and the purpose if their determination is currently unknown.…”

“…The ASFL region of ASNases follows the N-terminal 10-15 residues and extends over 20-25 amino acids [84]. It is alternatively called the N-terminal flexible loop [91] or the active site lid [88]. ASFL is the most dynamic element of an ASNase molecule, an observation that was made quite early [54,55,57].…”