The role of amino acid residues in the active site of a midgut microvillaraminopeptidase from the beetle Tenebrio molitor

Aminopeptidases are major enzymes in the midgut microvillar membranes of mo st insects and are targets of insecticidal Bacillus thuringiensis crystal d elta-endotoxins. Sequence analysis and substrate specificity studies showed that these enzymes resemble mammalian aminopeptidase N, although informati on on the organization of their active site is lacking. The effect of pH at different temperatures on the kinetic parameters of Tenebrio molitor (Cole optera) larval aminopeptidase showed that enzyme catalysis depend on a depr otonated (pK 7.6; Delta H degrees(ion), 7.6 kJ/mol) and a protonated (pK 8. 2; Delta H degrees(ion), 16.8 kJ/mol) group. 1-Ethyl-3-(3-dimethylaminoprop yl) carbodiimide and diethylpyrocarbonate inactivate the enzyme by modifyin g 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 af fecting its V-max by modifying a phenol group. The presence of a competitiv e inhibitor decrease the inactivation reaction rates in all these cases. ED TA inactivation of the aminopeptidase is affected by pH and temperature sug gesting the involvement in metal binding of at least one deprotonated imida zole group (pK 5.8, Delta H 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 substr ate binding relies in one phenol and one carboxylate groups. The insect ami nopeptidase shares common features with mammalian aminopeptidase N, althoug h differing in details of substrate binding and in residues directly involv ed in catalysis. (C) 2000 Elsevier Science B.V. All rights reserved.