Structural biology: Part 2 - Mechanism of Inverting Glycosidases

Exoglycosidases are glycosyl hydrolases that generally cleave a glycoside bond between a non-reducing terminal monosaccharide of a sugar polymer substrate and the adjoining monosaccharide. There is a specific nomenclature for the parts of the substrate in the cleavage reaction. The non-reducing terminal monosaccharide is called the GLYCONE, because it contributes the anomeric carbon to the glycosidic center in the acetal bond. The adjoining monosaccharide (and the remainder of the oligosaccharide substrate) is termed the AGLYCONE, and is effectively the leaving group in the catalytic mechanism. For example, consider the cleavage of a trisaccharide of Mana1,2Mana1,3Man-aMe to Man and Mana1,3Man-aMe. In this reaction the following parts of the structure are the glycone and aglycone:

Mana1,2Mana1,3Man-aMe -> Man + Mana1,3Man-aMe

red =GLYCONE blue=AGLYCONE

Cleavage of the glycoside bond can occur by one of two different types of general acid/general base mechanisms. Each of the two mechanisms has a unique stereochemical outcome resulting in release of a glycone with either retention or inversion of the original anomeric configuration. For example:

Man-a-X -> Man-a-OH + X <----retaining mechanism
Man-a-X -> Man-b-OH + X <----inverting mechanism

The two different stereochemical outcomes result from distinctive mechanisms of glycoside bond cleavage that are intrinsic to the geometry and stereochemistry of the enzyme active site. Thus an enzyme will either cleave with an inverting mechanism or a retaining mechanism, but not both. Enzymes that are related in sequence and structure generally all catalyze bond cleavage by a similar mechanism.

Both inverting and retaining mechanisms employ a pair of catalytic residues: a proton donor and a nucleophile/base. In both classes of mechanism the position of the proton donor is within hydrogen bonding distance from the glycosidic oxygen. In retaining enzymes the second catalytic residue is a nucleophilic carboxylate group that acts in a double displacement reaction, either as an ion pair stabilizing a transient oxonium ion transition state (i.e. HEW lysozyme) or a nucleophile/leaving group for a transient covalent intermediate (i.e. e-e b-glucosidases). In inverting enzymes a water molecule is used as a nucleophile in a single step reaction following activation by a carboxylate acting as a catalytic base.

Retaining Mechanism of Glycoside Bond Cleavge (Ion pair with oxocarbonium intermediate)

Retaining Mechanism of Glycoside Bond Cleavage (Covalent Intermediate)

Figure showing a Retaining Mechanism
(Click on the figure to enlarge)

A figure showing an Inverting Mechanism (Click on the figure to enlarge)

Inverting Mechanism of Glycoside Bond Cleavage

A classical example of an inverting glycosidase mechanism is glucoamylase from Aspergillus awamori. This enzyme cleaves a1,4 linked glucose residues from non-reducing terminus of starch and malto-oligosaccharides with the release of the glycone product as glucose-b-OH. The mechanism of catalysis has been investigated both by enzymatic approaches as well as structural studies in the presence of deoxyglucose aglycone analogs (1-deoxynojirimycin) and uncleavable maltooligosaccharides. The figure below to the left shows a ribbon structure of glucoamylase in the presence of each of the substrate analogs. The structure on the right shows the positioning of the two catalytic carboxylates in the active site of the enzyme relative to the glycone and aglycone substrates. Glu179 acts as the catalytic acid by protonating the glycosidic oxygen. Glu 400 acts as the catalytic base by abstracting a proton from the water nucleophile. The water is then activated to attack the glycosidic carbon from the bottom side and result in the elimination of the glycosidic oxygen and inversion of the configuration at the anomeric center to form a b-linked hydroxyl. An animation of the structure and the mechanism can be seen at the following GLUCOAMYLASE MECHANISM LINK.

Ribbon structures of glucoamylase in the presence of substrate analogs (click the figure to enlarge)

Two different views of the catalytic residues involved in catalysis by glucoamylase (click the figure to enlarge)

Part 3: Structure of human ER Mannosidase I