As shown in PART 3 of the Structural biology section, the potential catalytic residues in the active site of ER mannosidase I are in a highly unusual geometry for an inverting glycosidase (6). In contrast to glucoamylase, the classical inverting glycosidase (see PART 2), the structure of human ER mannosidase I is unusual in several respects:

1) Ca⁺² is directly involved in substrate binding to the enzyme.

2) The conformation of the inhibitors, presumably mimicking the conformation of the glycone, is in a highly unusual, and energetically unfavorable, ¹C₄"all-axial" conformation.

3) there do not appear to be appropriately positioned acidic amino acid side chains in proximity to the glycosidic bond to act in proton donation to the glycosidic oxygen.

The figures below show the position of the only three potential catalytic amino acids in proximity to the glycosidic bond, as derived from the human ER mannosidase I coordinate file ( E330, D463, and E599). These residues are shown in wireframe in CPK colors. The carbonyl oxygens are in spacefill and are colored red. The position of 1-deoxymannojirimycin in the active site of ER mannosidase I is also shown in wireframe display in CPK colors with a green spacefill at C1 equivalent to the glycosidic carbon. Figure 1 on the left shows that the water adjacent to Glu330 is on the wrong side of the plane of the sugar ring for the nucleophilic attack, and therefore Glu330 is not likely to be the catalytic base. In contrast, the only water molecule on the apppropriate side of the sugar ring (shown in green spacefill in Fig 1) is within hydrogen bonding distance to Glu599 and is also involved in the coordination of the Ca⁺² (magenta spacefill). This would be an unusual role for a protein-bound water, both acting in coordination to a divalent as well as actiing as a catalytic nucleophile. THE RESIDUES AND GEOMETRIES OF THE MOLECULES IN THE ACTIVE SITE CAN ALSO BE SEEN IN A CHIME ANIMATION.

Figure 1: Active site residues in ER mannosidase I (Click on the figure to enlarge)

Figure 2: Hypothetical mechanism of action for ER mannosidase I (Click on the figure to enlarge)

The position of the aglycone in the active site is shown in Figure 2 above (AND IN A CHIME ANIMATION OF THE STRUCTURE), and was generating a superimposition oligosaccharide aglycone from the yeast ER mannosidase I active site into the active site of the human ER mannosidase I (containing 1-deoxymannojirimycin). The oligosaccharide from the yeast enzyme structure is shown in yellow. The figure shows that the position of the O2' hydroxyl of the aglycone is whithin the expected proximity of the C1 of the glycone mimic, 1-deoxymannojirimycin. In addition, this would position the O3' and O4' hydroxyls in hydrogen bonding distance from Asp643 to stabilize the aglycone substrate interaction. This structure would appear to suggest that Glu599 acts as the catalytic base activating the bridging water for nucleophilic attack to the C1 carbon of the glycone. The question of the catalytic base in the enzymatic mechanism is even less certain. Normally a catalytic carboxylate would be expected to be within hydrogen bonding distance to the glycosidic oxygen. The only residue in proximity to the glycosidic oxygen is Glu330, but the distance is greater than would be expected for functioning as a catalytic acid (3.9A in the human enzyme and 4.3A in the yeast enzyme).

Two hypotheses have been proposed for the catalytic mechanism of ER mannosidase I (6). While both mechanisms invoke Glu599 as the catalytic base, in the first hypothesis the protonation occurs by the action of Glu330, possibly by a slightly different conformation than those in the crystal structure to account for their more direct proximity to the glycosidic bond. In the second hypothesis the substrate glycosidic oxygen interacts indirectly by a water molecule, possibly linked to an carboxylate amino acid (i.e. Glu330). The goals of future studies are to employ uncleavable substrate analogs and enzyme molecules containing mutations in the active site to trap uncleaved substrates in the active site. These studies, in addition to ongoing kinetic and binding studies should allow a more complete understanding of the mechanism of this highly unusual glycosylhydrolase.

Back to the Introduction