title: The role of amino acid residues in the active site of a midgut microvillar aminopeptidase from the beetle Tenebrio molitor.
abstract: Aminopeptidases are major enzymes in the midgut microvillar membranes of most insects and are targets of insecticidal Bacillus thuringiensis crystal delta-endotoxins. Sequence analysis and substrate specificity studies showed that these enzymes resemble mammalian aminopeptidase N, although information on the organization of their active site is lacking. The effect of pH at different temperatures on the kinetic parameters of Tenebrio molitor (Coleoptera) larval aminopeptidase showed that enzyme catalysis depend on a deprotonated (pK 7.6; DeltaH degrees (ion), 7.6 kJ/mol) and a protonated (pK 8.2; DeltaH degrees (ion), 16.8 kJ/mol) group. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and diethylpyrocarbonate inactivate the enzyme by modifying a pK 5.8 carboxylate and a imidazole group, respectively, with a reaction order around 1. Tetranitromethane changes the K(m) of the enzyme without affecting its V(max) by modifying a phenol group. The presence of a competitive inhibitor decrease the inactivation reaction rates in all these cases. EDTA inactivation of the aminopeptidase is affected by pH and temperature suggesting the involvement in metal binding of at least one deprotonated imidazole group (pK 5.8, DeltaH degrees (ion), 20 kJ/mol). The data support the hypothesis that T. molitor aminopeptidase catalysis depends on a catalytic metal and on a carboxylate and a protonated imidazole group, whereas substrate binding relies in one phenol and one carboxylate groups. The insect aminopeptidase shares common features with mammalian aminopeptidase N, although differing in details of substrate binding and in residues directly involved in catalysis.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970, São Paulo, Brazil.
authors.forename: P T
authors.initials: PT
authors.lastname: Cristofoletti
authors.forename: W R
authors.initials: WR
authors.lastname: Terra
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D013696:Temperature; D013712:Tenebrio; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D002614:Chelating Agents; D000626:Aminopeptidases