XAO

XAO

Xanthine oxidase is an enzyme with low specificity, which can not only catalyze the formation of xanthine from hypoxanthine to uric acid, but also directly catalyze the formation of uric acid from xanthine. Sulfide and flavinase of FAD are found in milk, animals (especially the liver and kidney of birds), insects and bacteria. Xanthine oxidase has a molecular weight of tens of thousands to tens of thousands, and a wide substrate specificity. In addition to purine derivatives as electron donors, pteridine derivatives and aldehydes (generating carboxylic acids) can also be electron donors. Hydroxyl compounds are formed, and oxygen atoms originate from water. There are many electron acceptors for xanthine oxidase, including molecular oxygen, nitrate, benzoquinone, nitro compounds, NAD, ferredoxin, etc., but it varies according to the source of the enzyme. Peroxide is generated during the reaction, causing a chain reaction.

Figure 1. Protein structure of XAO.

Structure

The molecular weight of xanthine oxidase is relatively large, about 270,000, and contains two molecules of FAD, two molybdenum atoms and eight iron atoms. The molybdenum in the enzyme exists in the form of molybdopterin cofactor and is the active site of the enzyme. Iron atoms are part of the [2Fe-2S] ferredoxin iron-sulfur clusters and participate in electron transfer reactions. Xanthine oxidase is a flavin protease that exists in various organisms and can catalyze the formation of uric acid from purine substrates in the body. The crystal structure of xanthine oxidase has been resolved. It is composed of 1,330 amino acids, and its amino acid sequence has 90% homology between mice and humans. It is composed of two completely symmetrical structural units, each structure unit is 145 ku, and its catalytic center includes a molybdopterin center, two iron-sulfur centers, and a flavin adenine dinucleotide, of which the molybopterin center is a key site for xanthine to uric acid catalyzed by xanthine oxidase point.

Catalytic mechanism

The molybdenum atom of the molybopterin cofactor in the active site of xanthine oxidase is additionally connected to a terminal oxygen, multiple sulfur atoms, and a terminal hydroxyl group. In the reaction of xanthine to uric acid, the oxygen on the molybdenum is first transferred to the xanthine molecule. Then, the water molecule is added to the active intermediate to regenerate the active molybdenum center. Similar to other known molybdenum-containing redox enzymes, the newly introduced oxygen atoms in the product come from oxygen in water molecules, not oxygen molecules.

Application

1. Participate in the catabolism of nucleic acids in the body

Human uric acid is mainly decomposed by nucleic acids and other purine compounds that are metabolized by cells and purines in food by the action of enzymes. Uric acid is the final product of the catabolism of adenine and guanine, the constituent components of nucleic acid, in the human body. Hypoxanthine and xanthine are direct precursors of uric acid. Under the action of xanthine oxidase, hypoxanthine is oxidized to xanthine, and xanthine is oxidized to uric acid.

2. Promote the absorption and transfer of iron

In small intestinal mucosal cells, xanthine oxidase oxidizes ferrous ions absorbed from food into ferric ions, which are absorbed by plasma transferrin and transported to various tissues like blood.

3. Test the activity of SOD (superoxide dismutase)

In the presence of oxygen, xanthine oxidase can catalyze the oxidation of xanthine to generate uric acid and oxygen free radicals. The generated superoxide anion free radical, the nitrite formed by the oxidation of hydroxylamine shows a purple-red color under the action of the developer, and its absorbance is measured with a visible light spectrophotometer. When the tested sample contains SOD, it has a specific inhibitory effect on superoxide anion free radicals, which reduces the formation of nitrite and the absorbance of the measuring tube is low, thereby calculating the SOD activity in the tested sample.

Inhibitor

The structure of xanthine oxidase is very complex, so many substances can inhibit its activity. Substances such as purines, pyrimidines and other heterocycles can compete with the substrate to bind to the active site of the enzyme, called competitive inhibitors; compounds such as arsenite, cyanide and methanol can interact with molybdenum atoms to the enzyme is inactivated. Other inhibitors include phosphate, imidazole, sodium, potassium chloride, benzoate, borate, copper, ascorbic acid, and dinitrophenol. Among them, the inhibition of ascorbic acid on xanthine oxidase is an indirect effect. Milk contains divalent copper ions, and ascorbic acid can reduce Cu²⁺ to Cu⁺, which is a strong inhibitor of the enzyme.

Reference

1. Bonini MG. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate. The Journal of Biological Chemistry. 2004, 279 (50): 51836–43.