COMPARATIVE BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF TWO E. coli L-ASPARAGINASE II (EcA2) VARIANTS

INTRODUCTION: Escherichia coli l-asparaginase II (EcA2) is essential for the treatment of acute lymphoblastic leukemia (ALL), the most common childhood cancer. This enzyme catalyzes the hydrolysis of l-asparagine or l-glutamine into ammonia and l-aspartate or l-glutamate. Several biosimilars of l-asparaginase (EcA2) have been developed, but despite rigorous approval criteria, variations in plasma activity and therapeutic efficacy persist between different EcA2 preparations, often leading to reports of substandard products. AIMS: To characterize two EcA2s from different E. coli strains: one from the K12 strain, and is considered the reference sequence in the Uniprot and many of the scientific literature, and the other from the BL21(DE3) strain, equivalent to that used in the originator biopharmaceutical product Elspar^®, which has four point mutations (4M) compared to K12. MATERIAL AND METHODS: EcA2 variants were expressed in E. coli strain BL21 (DE3) and purified by tangential flow filtration and chromatography. Enzymatic activity was quantified by direct asparagine absorptiometry and NMR analysis. Conformational stability was assessed by circular dichroism (CD), intrinsic fluorescence, and NMR spectroscopy. Cytotoxicity was evaluated in lymphoid leukemia cells (CCRF-CEM). RESULTS: EcA2 from both E. coli strains showed similar global conformation and thermal stability, as investigated by CD, NMR, and fluorescence. However, EcA2-4M showed more than twice the specific activity in both l-asparagine hydrolysis and in human lymphoblastic cells compared to EcA2-K12. EcA2-K12 demonstrates 4-fold higher specificity for l-asparagine relative to l-glutamine, considering its k_cat, but similar K_M for each amino acid. Interestingly, EcA2-K12 has 3-fold lower affinity for l-aspartate, linked to the reduced stabilization of its N-terminal active site loop. We solved the 3D structures of both variants by X-ray crystallography, and normal mode analysis revealed broader conformational changes in the active site of EcA2-K12, indicating that this variant has lower activity due to the greater dynamics of the N-terminal active site loop. CONCLUSION: Our results highlight that EcA2-4M exhibits a similar overall conformation to EcA2-K12, albeit with higher activity through more efficient substrate-to-product conversion. However, EcA2-K12 is a beneficial alternative or complement to existing EcA2-4M therapeutic regimens due to its greater specificity for l-asparagine, which is of fundamental importance for the treatment of ALL.