Bases moleculares do efeito do pH na atividas catalítica de duas lisozimas digestivas de Musca domestica (Diptera) / Molecular basis of the pH effect on the catalytic activity of two digestive lysozymes from Musca domestica (Diptera)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.

ASSUNTO(S)

biologia molecular digestão animal ph optimum musca domestica proteins (structure) musca domestica insects (enzymology; study) lisozima lysozyme proteínas (estrutura) lisozima digestiva digestive lysozyme ph ótimo insetos (enzimologia; estudo) molecular biology

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