Análise de uma L-asparaginase humana: a busca por determinantes estruturais importantes para o aumento de sua eficiência catalítica

Abstract

One of the drugs most used to treat acute lymphoid leukemia (ALL) is the enzyme L- asparaginase. However, the main commercially available L-asparaginases (E.C. 3.5.1.1), which come from Escherichia coli and Erwinia chrysanthemi, have triggered side effects in different patients. Therefore, humanized L-asparaginases have been sought, because they have lower immunogenic potential and may eliminate the side effects. hASNase1 is a human L-asparaginase that has lower kinetic parameters, like specificity constant kcat/Km, than the main commercially available L-Asparaginases. From previous works with hASNase1, it was hypothesized that residues from the active site´s vicinities of the active site of this enzyme may be involved in the fixation of its substrate, L-Asparagine (Asn), in this region. This behavior may be hindering the access of the substrate to the active site of hASNase1, which may be responsible for the lower catalytic efficiency of this enzyme. The goal of this work was to use MD simulation trajectories to analyze the Asn output of the catalytic site of hASNase1, in order to identify residues that interact with this substrate and maintain it in the adjacent region to this site. Interatomic Interaction Potentials (IIP) were investigated to indicate the placement and setting of Asn in the vicinity of the active site of hASNase1. We also investigated the formation of Hydrogen Bond (HB) between Asn and adjacent residues to the active site of hASNase1. It has been observed that Asn is immobilized by residues located outside the catalytic site of the enzyme. Two residues that showed attractive interaction with Asn were identified. Because of this, these residues were related to the setting of the substrate in the vicinity of the site. A low frequency of HB formation was observed between Asn and residues located in the vicinity of the active site of hASNase1. Therefore, evidences were generated which link the two residues identified with the lower catalytic efficiency of hASNase1. Thus, these residues have become strong candidates for mutations in order to obtain variants of hASNase1 that can be marketed as biopharmaceuticals against ALL.