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

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 micro villar proteins. AP, after being solubilized in detergent or released by pa pain, was purified to homogeneity. The enzyme is a glycoprotein rich in man nose residues. N-terminal sequencing of papain and detergent forms of AP re sulted in the same sequence containing the common motif YRLP. These and oth er data, which included partition in Triton X-114 and incubation with glyco syl-phosphatidylinositol (GPI)specific phospholipase C and GPI-specific pho spholipase D suggest that AP (Mr 90 000) is inserted into the microvillar m embranes 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 ami no acid residue of substrates, although it does not hydrolyze acidic aminoa cyl-peptides, thus resembling mammalian aminopeptidase N (EC 3.4.11.2). k(c at)/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' an d S-2' are pockets able to bind bulky aminoacyl residues. This hypothesis a grees with the fact that amastatin is a stronger inhibitor of AP than besta tin. 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. (C) 1999 Elsevie r Science Ltd. All rights reserved.