Characterization of intestinal trehalase of the larvae Tenebrio molitor and cloning of cDNA that encodes

Author(s)

Ana Gilhema Gomez Duran

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2005-10-14

Examining board members

Clelia Ferreira Terra; Ivo Lebrun; Maria Teresa Machini de Miranda; Carla Columbano de Oliveira; Ivarne Luis dos Santos Tersariol

• Advisor: Clelia Ferreira Terra

Abstract

Intestinal trehalase was purified from Tenebrio molitor larvae after three chromatographic steps (hidrophobic, interaction, ion exchange chromatography and gel filtration). The pure enzyme has an specific activity of 16.5 U/mg, molecular mass of 58 kDa, optimum pH of 5.3 and Km of 0.43 ± 0.03 mM. Amygdalin (Ki = 0.22 mM), prunasin (Ki = 0.43 mM), phlorizin (Ki = 0.50 mM), phloretin (Ki = 0.008 mM), methyl-α-mannoside (Ki = 43 mM), salicin (Ki = 186 mM), and glucone-δ-lactone (Ki = 1.4 mM) are competitive inhibitors of the enzyme. Mandelonitrile (the aglycon of the glucosides amygdalin and prunasin) is a non-competitive mixed-type inhibitor (Ki = 3.8 mM and α = 1.5). Gentiobiose, methyl-α-glucoside, 1,10 phenanthroline and Tris in concentrations of 10 mM, 200 mM, 4 mM, and 264 mM, respectively, were unable to inhibit the enzyme. We designed a model for trehalase active site, taking into account inhibition and multiple inhibition experiments plus protection afforded by competitive inhibitors against the chemical modification of amino acid residues. The site has two assimetric subsites for glucose binding. Phloretin binds to subsite II and methyl-α-mannoside and glucone-δ-lactone bind to subsite I. In this subsite, one His residue modulates the p(Ka of the carboxylate group that acts as a nucleophile in catalysis. The carboxylate and one Arg residue, that acts as a proton donor, are placed in the region between the two subsites. Using RT-PCR techniques, the cDNA coding for T. molitor intestinal trehalase was cloned. From the sequence, we can suppose that the enzyme is soluble and calculate that the molecular mass of the protein would be 61 kDa. T. molitor trehalase can be classified as a member of family 37 of glycoside hydrolases. No member of this family has their catalytical groups nor its 3D structure known. (AU)