The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B

Abstract

Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases.

Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein inSaccharomyces cerevisiae

“…We also observed that the tyrosine residue involved in the catalytic mechanism and generally located 80–90 residues after the HEXXHX 18 E motif in M1 aminopeptidases was conserved in Tma108 ( 40 ). The (G/A/H/V)(G/A)MEN motif, found in 77% of the M1 family proteins and involved in substrate binding and recognition ( 41 ), was also present in Tma108. Only the 293 M in the first position of the motif (MAMEN) is not congruent with the defined signature.…”

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases

“…In an “open” form of the protein, this residue undergoes a 6 Å shift away from the zinc ion, whereas in a closed form, this Tyr residue is proposed to both assist in the activation of the peptide bond and in the stabilization of the tetrahedral intermediate during the proteolytic catalytic cycle [ 21 , 22 ]. It has been shown, by site directed mutagenesis, that mutation of this Tyr residue into Phe completely abolished the enzyme activity [ 82 , 83 ].…”

The effects of curcumin, mangiferin, resveratrol and other natural plant products on aminopeptidase B activity

“…In Ap-B, similar hydrogen bonds should not be established, owing to a Phe residue in position 297 instead of a tyrosine (Fig. 1-B, [26]). Thus, trans-resveratrol could interact differently with the Ap-B structure, which could explain why resveratrol has no significant effect on its activity.…”