Simulações de dinâmica molecular de L-asparaginases: estudo comparativo entre uma L-asparaginase humana, bacteriana e do porquinho da Índia

Abstract

L-Asparaginase is an enzyme that catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. This enzyme is used in the treatment of some types of cancer, especially ALL, along with other drugs. Commercial enzymes are derived from bacteria, for example, Escherichia coli (EcII), and they cause adverse reactions during treatment. A human enzyme could be a non-immunogenic substitute; however, its kinetic properties are not as efficient. The human enzyme studied in this work is hASNase1, which belongs in the N-terminal domain of the 60kDa-lysophospholipase protein, and exhibits allosterism. There is no 3D structure available for it and, although hASNase1 displays a high structural identity (>70%) with gpASNase1 from Cavia porcellus, they do not share the same catalytic efficiency. However, gpASNase1 has similar kinetic properties to EcII. The aim of this study was to model hASNase1 and compare its structure with that of gpASNase1 and EcII using Molecular Dynamics (MD) in order to understand structural differences in solution that may explain the different kinetic properties. hASNase1 was submitted to MD, as well as gpASNase1 (PDB code: 4R8K) and EcII (PDB code: 3ECA), in aqueous medium, containing Asn bound or not, to the catalytic and/or allosteric sites. The structural and thermodynamic properties were evaluated by analysis of the trajectory. The results of MD revealed that hASNase1 is a dimer of dimers and its structure has a gap in the center between the monomers as well as gpASNase1. The interaction potential between Asn and catalytic site residues showed greater freedom of substrate orientation in hASNase1 and gpASNase1 than in EcII. The loop1 in hASNase1 has a constitution of residues that makes it more susceptible to deformations and movements. Loop2, on the other hand, has an -helix in hASNase1, which leaves the position of Tyr308 more fixed at the site than in gpASNase1 and it may cause a difference in catalytic efficiency. Analysis of Lys188 movement showed the effect of positive cooperativity of hASNase1and it was found that the presence of Asn at the allosteric site stabilizes Lys188 for the maintenance of the triad. It also helps to stabilize the movement of the Loop1. In conclusion, despite the structural similarity between hASNase1 and gpASNase1, there are dissimilarities, besides allosterism, which may explain the different kinetic properties.