EXPLORING CONFORMATIONAL STABILITY AND ENZYMATIC ACTIVITY IN VARIANT FORMS OF L-ASPARAGINASE TYPE 2 FROM Escherichia coli

INTRODUCTION: L-asparaginase type 2 (EcA2) from Escherichia coli is a crucial biopharmaceutical for treating Acute Lymphoblastic Leukemia, functioning by catalyzing the hydrolysis of l-asparagine into aspartate and ammonia. Notably, it has been described disparities between the amino acid sequences of EcA2 in medications utilized in Brazil and the standard reference sequence from the E. coli K12 strain. AIMS: This study characterizes two EcA2 variants: one with the canonical K12 sequence and another from the biopharmaceutical Aginasa®, which has four point mutations (4M) compared to K12. MATERIAL AND METHODS: The EcA2 variants were expressed in E. coli BL21 (DE3) strain and purified by tangential flow filtration and chromatography. Enzymatic activity was quantified via direct asparagine absorptiometry and NMR analysis. Conformational stability was assessed using circular dichroism (CD), intrinsic fluorescence, and NMR spectroscopy. Cytotoxicity was evaluated in lymphoid leukemia cells (CCRF-CEM). RESULTS: Circular dichroism spectroscopy, intrinsic tryptophan fluorescence, and NMR demonstrated similar global conformation and thermal stability between the two variants. However, the 4M variant exhibited a threefold increase in turnover rate for l-asparagine and doubled toxicity against human lymphoblast cell lines compared to K12. Both enzymes had a significantly lower K_M for l-asparagine compared to l-glutamine, but 4M’s k_cat was three times higher than K12’s. NMR and intrinsic fluorescence revealed that the 4M variant had a threefold higher affinity for aspartate compared to K12. Consequently, 4M exhibited enhanced conformational stability in the N-terminal loop, which is part of the active site. CONCLUSION: Our results highlight the complexity nature of the catalytic mechanism of L-asparaginase, showing that the mutations in 4M lead to higher activity through more efficient substrate-to-product conversion.