Caracterização bioquímica e cinética de L-asparaginases bacterianas e avaliação de seus potenciais citotóxicos

Abstract

L-asparaginase (L-ASNase) is an enzyme that hydrolyzes L-asparagine, generating L-aspartate and ammonia. It is known to have two enzymatic isoforms, being named type I and type II. Type II L-ASNase is widely used in chemotherapeutic routines against Acute Lymphoid Leukemia as an adjuvant antineoplastic agent whose mechanism of action is based on the depletion of serum L-asparagine, a non-essential amino acid important for leukemia cells proliferation. The major commercial oncoterapic L-ASNases are obtained from the bacteria Escherichia coli and Erwinia chrysanthemi. However, adverse effects and hypersensitivity are attributed to its use due to the presence of glutaminase activity. Therefore, strategies aimed at characterizing and studying the viability of L-asparaginases may aid in the development of biomolecules with improved therapeutic properties in order to promote a more effective and safer treatment. The present work aimed to characterize different bacterial L-asparaginases specifically that of Escherichia coli, called L-ASNase B, and those of Bacillus subtilis, L-ASNases A and Z, and determine their cytotoxic potentials in different leukemic cell lines. The enzymatic activities were evaluated by the Nessler method, whereas for the cytotoxicity and cell viability assays the MTT reagent was used. Optimum pH and temperature reaction conditions of L-ASNases B and Z were described previously by the group. The optimum pH for L-ASNase B was 8.5 and the optimum temperature was 60 °C, while for L-ASNase Z the optimum pH was 7.0 and the optimum temperature was 50 °C. The characterization of L-ASNase A was carried out and determined obtaining as optimum pH 7.0, and as optimum temperature 40 °C. Furthermore, when using L-glutamine as a substrate, L-ASNase B was shown to have a higher glutaminase activity than L-ASNase Z. The kinetic parameters of the enzyme L-ASNase B were 3 mM for Km and 168.2 μmol/min/mg for Vmáx, compared to the L-asparagine substrate. For L-ASNase Z, the values were 1.43 mM and 90.0 μmol/min/mg for Km and Vmáx, respectively, also compared to L-asparagine. In relation to the cytotoxicity assay performed in the HL-60 cell line, L-ASNase B had an IC50 of 0.83 μg/mL and for L-ASNase Z the IC50 was 1.53 μg/mL. The enzymes characterized have potential application in the oncological area, demonstrating the promising approaches for the optimization of the biocatalysts from the presented results, in a way that opens up increasingly perspectives for an efficient therapy to leukemic patients.