Catalytic mechanism of human alpha-galactosidase

Abstract

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a (1)S(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

FIGURE 1.

α-GAL reaction and trapping stages for crystallographic analysis. A, double displacement reaction mechanism in human α-GAL. Asp-170 acts as the nucleophile, and Asp-231 acts as an acid and then a base over the course of the reaction cycle. B, structures of the different stages in the catalytic cycle. Empty enzyme (blue) required a cryoprotectant sterically excluded from the active site. The substrate-bound structure (green) resulted from deletion of the active site nucleophile, followed by addition of a disaccharide substrate. The covalent intermediate (yellow) used a difluoro-substituted galactoside to slow the second stage of the reaction. The product-bound structure (red) resulted from product inhibition of the enzyme. The color scheme is maintained throughout.

FIGURE 2.

Ligand density and interactions. A–D, first and second columns show side and top views of the electron density for the ligand in the four different structures. The third column shows the interactions around the ligand in the active site, where red lines represent hydrogen bonds and blue lines represent van der Waals interactions. Empty (A), substrate-bound (B), covalent intermediate (C), and product-bound structures (D) are shown, respectively. The electron density corresponds to a σ_A-weighted 2F_o-F_c total omit map calculated in SFCHECK (25), contoured at 1.5σ in A and 2.0σ in B–D, with a cover radius drawn around residues and/or waters in the active site.

FIGURE 3.

Covalent intermediate and ligand deformation. A, reaction mechanism for the TNP-2,2-di-F-α-Gal substrate. The first step of the reaction remains fast because of the good leaving group, but the second step slows, allowing for trapping of the covalent intermediate species. B–D, close-up views of the substrate-bound, covalent intermediate, and product-bound ligands. The catalytic nucleophile Asp-170, the catalytic acid/base Asp-231, and the conserved Trp-47 are labeled. The ligand conformation changes from ⁴C₁ to ¹S₃ to ⁴C₁ geometry over the course of the reaction. The modeled transition states in ⁴H₃ half-chair conformations are shown in the insets.

FIGURE 4.

The second ligand-binding site in human α-GAL. The second ligand-binding site is centered on Tyr-329, at the interface between domain 1 and domain 2 of the structure. A surface drawn around all the atoms reveals a pocket that is selective for β-galactose. The electron density from the galactose-soaked crystal shows a σ_A-weighted 2F_o-F_c total omit map calculated in SFCHECK, contoured at 1.1σ with a cover radius drawn around the ligand. Residues Glu-251, Asp-255, Tyr-329, and Lys-374 are shown.