Specificity, anchoring, and subsites in the active center of a microvillar aminopeptidase purified from Tenebrio molitor (Coleoptera) midgut cells

Abstract

There is a single membrane-bound aminopeptidase (AP) in Tenebrio molitor L. larval midguts with a pH optimum of 8.0. This enzyme is restricted to the posterior third of the midgut, where it accounts for about 55% of the microvillar proteins. AP, after being solubilized in detergent or released by papain, was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues. N-terminal sequencing of papain and detergent forms of AP resulted in the same sequence containing the common motif YRLP. These and other data, which included partition in Triton X-114 and incubation with glycosyl-phosphatidylinositol (GPI)-specific phospholipase C and GPI-specific phospholipase D suggest that AP (Mr 90 000) is inserted into the microvillar membranes by a C-terminal anchor, which is a peptide or a papain — released protected GPI anchor. AP has a broad specificity towards the N-terminal amino acid residue of substrates, although it does not hydrolyze acidic aminoacyl-peptides, thus resembling mammalian aminopeptidase N (EC 3.4.11.2). k cat/K m ratios obtained for different di-, tri-, tetra-, and pentapeptides suggest that there are four subsites in AP, and that subsites S 1, S 1′ and S 2′ are pockets able to bind bulky aminoacyl residues. This hypothesis agrees with the fact that amastatin is a stronger inhibitor of AP than bestatin. Amastatin is a slow, tight-binding inhibitor of AP. Bestatin binds in a rapidly reversible mode in S 1′ and S 2′ subsites of AP.