Glucose Isomerase

Glucose isomerase (GI, EC 5.3.1.5), also known as xylose isomerase, D-xylose isomerase, D-xylose keto isomerase, and D-xylose ketol-isomerase, is an isomerase that isomerizes aldose, such as D-xylose, D-glucose, D-ribose, etc., to the corresponding ketose. The systematic name of this enzyme class is D-xylose aldose-ketose-isomerase. Glucose isomerase has a wide range of sources, including microorganisms, such as bacteria, fungi and actinomycetes, as well as plants and animals. Glucose isomerase is a key enzyme in the industrial production of high fructose corn syrup and fuel ethanol.

Enzymatic Properties

• Substrate specificity

Glucose isomerase has a wide range of substrates, in addition to D-glucose and D-xylose, glucose isomerase can also catalyze substrates such as D-ribose, L-arabinose, L-rhamnose, D-allose, deoxyglucose, etc. However, glucose isomerase can only catalyze the conversion of the alpha-optical isomer of D-glucose or D-xylose, but not the beta-optical isomer.

• Optimum Temperature and pH

The optimum pH of glucose isomerase is usually slightly alkaline, between 7.0 and 9.0. Under the acidic conditions, the enzyme activity of most species is very low. The optimal reaction temperature of glucose isomerase is generally 70-80°C.

• Metal Ion Requirement and Inhibitors

GI requires a divalent cation such as Mg²⁺, Co²⁺, or Mn²⁺, or a combination of these cations, for maximum activity. Although both Mg²⁺ and Co²⁺ are essential for activity, they play differential roles. While Mg²⁺ is superior to Co²⁺ as an activator, Co²⁺ is responsible for stabilization of the enzyme by holding the ordered conformation, especially the quaternary structure of the enzyme.

Structure

Although glucose isomerases from different species have certain differences in the primary structure of proteins, they have similar spatial structures. Glucose isomerases are all non-glycoproteins and are generally present in the form of tetramers or dimers. Its subunit monomer has a molecular mass of 19-52 kD. The tetrameric subunits are bound by non-covalent bonds without disulfide bonds, the binding between dimers is stronger than the binding between subunits in dimers. The group of subunits is symmetrically distributed, and each subunit monomer is divided into two domains.

The N-terminal main domain consists of a "catalytic pocket" consisting of 8 strands of α/β helical folded structure, the inner layer consists of 8 parallel β-sheets, and the outer layer consists of 8 strands of α-helix alternately adjacent to the β-sheets. The peptide chain of the α-helix is antiparallel to the β-sheet, and the active center is located at the near C-terminus of the β-sheet. The small domain at the C-terminus is randomly coiled by a few α-helix into an irregular circular structure away from the N-terminus, which participates in the interaction between the subunits and the construction of the active center. The tetrameric glucose isomerase has four active centers, which are pocket-shaped, and the active center is located at the near C-terminus of the β-barrel of the subunit catalytic domain. Each active center consists of two adjacent subunits, containing two divalent metal ion binding sites, as well as conserved residues associated with substrate binding and catalytic processes.

Mechanism of Action

The catalytic mechanism of GI has been a subject of great interest to researchers. Earlier, GI was assumed to function in a manner similar to sugar phosphate isomerases and to follow the ene-diol mechanism. Recent studies have attributed the action of GI to a hydride shift mechanism. Knowledge of active-site configuration is a prerequisite for studying the structure-function relationship of the enzyme. Different approaches have been used to study the active site of GI and to delineate its mechanism of action. These include (i) chemical modification, (ii) X-ray crystallography, and (iii) isotope exchange. The main features of the mechanism proposed for GI are ring opening of the substrate, isomerization via a hydride shift from C-2 to C-1, and ring closure of the product.

Figure 1. Mechanism of action of GI. (a) cis-Enediol. (b) Proton transfer. (c) Hydride shift.
(Bhosale, S.H., et al. 1996)

In the isomerization of glucose, Histidine 53 is used to catalyze the proton transfer of O1 to O5. In the isomerization of xylose, crystal data has shown that xylose sugar binds to the enzyme in an open chain conformation. Metal 1 binds to O2 and O4, and once bound, metal 2 binds to O1 and O2 in the transition state, and these interactions along with a lysine residue help catalyze the hydride shift necessary for isomerization. The transition state consists of a high energy carbonium ion that is stabilized through all the metal interactions with the sugar substrate.

Production and Application

The cost of production of the enzyme is an important factor in evaluation of its suitability for industrial application. Intensive efforts have been made to optimize the fermentation parameters for the production of GI with a view to developing economically feasible technology. Research is focused on three major aspects: (i) improvement of the yields of GI, (ii) optimization of the fermentation medium with special reference to replacement of xylose by a cheaper substitute and elimination of requirement for Co21 ions, and (iii) immobilization of the enzyme. The most widely used application of this enzyme is in the conversion of glucose to fructose to produce high fructose corn syrup (HFCS). Xylose isomerase is one of the enzymes used by bacteria in nature to use cellulose as food and another focus on industrial and academic research, has been developing versions of xylose isomerase that could be useful in the production of biofuel.

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