**4. GPX**

**Figure 3.** The reaction cycles of catalase [35].

group and be involved in the enzyme reaction.

Catalase mainly used in industrial sectors such as textiles, pulp, and paper, their work environment often with high concentration of metal ions. Previous studies have elaborated

depends on the metal, concentration, the tissue, and species [38]. Lee et al. [39] compared several divalent metal ions on catalase‐peroxidase (KatG) activity, only the manganese ion revealed some inhibitory effects on the recombinant KatG activity, and EDTA could relieve partly inhibited activity. This implies that manganese may competitively bind to near the heme

), a process

that catalase can be inhibited by certain metal ions (including Cu2+, Zn2+, and Ag+

*3.2.2. Competitive inhibitor*

214 Enzyme Inhibitors and Activators

*3.2.2.1. Metal ions*

#### **4.1. The structure and function of GPX**

GPX is an important selenium‐containing enzyme which protects cells from lipid peroxide damage and H2O2. GSH‐Px widely existed in the body, there are eight family members: GPX1 is the most abundant selenoperoxidase and is ubiquitously expressed in almost all tissues; GPX2 expression is most prominent in the gastrointestinal tract cytoplasm; GPX3 is greatest expressed in the kidney, and also in various tissues, and is secreted into extracellular fluids as a glycoprotein; GPX4 is the only GPX enzyme that reduces phospholipid hydroperoxides, different with other members, it is not a tetramer, but a monomer; GPX6 was identified as a selenoprotein in the human genome by homology search. GPX1‐4 and GPX6 are selenium‐ containing protein, but GPX5 does not contain selenocysteine or Se in active site [41, 42]. More recently, GPX7 (NPGPx) and GPX8 were discovered, but detailed information about these two kinds of enzymes is little up to now [43].

Molecular weight of GSH‐PX in human red blood cell was 95,000 Da and that in bovine red blood cell was 83,000 Da, and they are all tetrameric selenoenzyme. A typical crystal structure of human glutathione peroxidase (2I3Y) is shown in **Figure 4**.

GSH‐PX contains one selenocysteine per subunit and selenocysteine plays an important role in catalyst degradation of lipid peroxide, a widely accepted mechanism was proposed as follow:

E CysSe H ROOH E CysSeOH ROH - + - + + ®- + (3)

$$\text{E}-\text{CysSeOH} + \text{GSH} \rightleftharpoons \text{E}-\text{CysSe} - \text{SG} + \text{H}\_2\text{O} \tag{4}$$

$$\text{E}-\text{CysSe}-\text{SG} + \text{GSH} \rightleftharpoons \text{E}-\text{CysSe}^{\bullet} + \text{GSSH} + \text{H}^{+} \tag{5}$$

**Figure 4.** Three‐dimensional structure of human glutathione peroxidase.

#### **4.2. The effect of selenium on GPX activity**

There are approximate 25 selenoproteins in humans, and selenium is an essential cofactor for these proteins, including the glutathione peroxidases. There is a complex relationship among utilization of selenium, GPX activity, and methylation. GPX synthesis utilizes selenium via selenocysteine and homocysteine is derived from *S*‐adenosylhomocysteine, and the latter was formed as a result of methylation reactions including methylation of selenium [44]. From the view of selenium forms, the inorganic forms (such as sodium selenate and sodium selenite) of selenium were more effective on increasing the GPX activity compared with selenomethio‐ nine [44]. To be attention, different members of GPXs response differently to selenium deficiency, a phenomenon called the "hierarchy of selenoproteins." This means that upon selenium deprivation, some proteins decline fast, whereas others remain synthesized until selenium becomes severely deficient [45]. The expression of GPX1 mRNA, protein, and its activity in tissues is more sensitive than other selenoperoxidases or selenoproteins [46].

#### **4.3. The effect of vitamin E on GPXs activity**

dl‐Alpha‐tocopherol (vitamin E) also is an antioxidant, but it is different from selenium acting on GPX directly, vitamin E plays its antioxidant function through combining free radical, named "chain‐breaking reaction." Vitamin E was considered to be the first line of defense against lipid peroxidation and free oxygen radicals that might suppress the enzymes, such as GPX. It seems that the sensitivity of GPXs was various. In vitamin E‐deficient rat brain microsomes, phospholipid hydroperoxide glutathione peroxidase activity was significantly decreased but GPX activity was not affected. And in liver homogenate, phospholipid hydro‐ peroxide glutathione peroxidase activity was approximately 20 times lower than that of GPX [47].

#### **4.4. Inhibitors of GPXs**

E CysSe H ROOH E CysSeOH ROH - + - + + ®- + (3)

**Figure 4.** Three‐dimensional structure of human glutathione peroxidase.

There are approximate 25 selenoproteins in humans, and selenium is an essential cofactor for these proteins, including the glutathione peroxidases. There is a complex relationship among utilization of selenium, GPX activity, and methylation. GPX synthesis utilizes selenium via selenocysteine and homocysteine is derived from *S*‐adenosylhomocysteine, and the latter was formed as a result of methylation reactions including methylation of selenium [44]. From the view of selenium forms, the inorganic forms (such as sodium selenate and sodium selenite) of

**4.2. The effect of selenium on GPX activity**

216 Enzyme Inhibitors and Activators

(4)

(5)

#### *4.4.1. Competitive inhibitors*

#### *4.4.1.1. Misonidazole*

GPX can combine to an electrophilic compound that might result in loss of its activity. More and more evidence shows that upregulation of the GPX system may serve to protect cancer cells from oxidative stress caused by anticancer drugs, thus block GPX that may help to treat cancer disease. A number of inhibitors of GPXs have been reported to use as therapeutics, such as thiol‐containing inhibitors that bind covalently to a selenium atom in the active site [48], nonthiol inhibitors misonidazole [49]. However, thiols tend to combine ubiquitous multivalent metal ions and are easily oxidized, thus leading to nonspecific interactions with proteins. Recently, acylhydrazones have been reported as potential inhibitors of bovine glutathione peroxidase [50]. These inhibitors overcome the disadvantages of thiol‐containing inhibitors, but the efficiency needs to be further improved.

#### *4.4.1.2. Penicillamine and its analogues*

d‐Penicillamine is a drug to chelate metals in tissue and promotes its excretion in the urine. d‐Penicillamine hydrochloride could competitively inhibit GSH‐PX, that means the concen‐ tration of hydrogen peroxide and reduced glutathione were inversely proportion [51].

l‐penicillamine hydantoin is an analogue of glutathione, but the acting configuration is different from d‐penicillamine hydrochloride. After treated with l‐penicillamine hydantoin, GPX activity was inhibited whatever the peroxide (H2O2, terl‐butyl hydroperoxide orcumene hydroperoxide) used as substrate of the reaction. In the presence of 100 μM l‐penicillamine hydantoin, the enzyme reactions catalyzed by glutathione peroxidase were inhibited, but neither glutathione transferases, nor glutathione reductase were affected by l‐penicillamine hydantoin [52].

#### *4.4.2. Noncompetitive inhibitors*

#### *4.4.2.1. dl‐Buthionine‐[S, R]‐sulfoximine*

dl‐Buthionine‐[*S, R*]‐sulfoximine (BSO) can be used as an inhibitor to estimate the scavenging efficiency of H2O2 after GPX inhibition. The inhibitory effect did not act on GPX directly, but through suppress the synthesis of GSH by inhibiting γ‐glutamylcysteine synthetase, that cause glutathione decreased sharply in many tissues, especially kidney, liver, and pancreas [53]. BSO has an obvious inhibitory effect, for example in human fibroblasts cells, after 500 μM BSO treated, the GSH levels decreased to154.0 ± 16.9 nmol/mg protein from 418.4 ± 13.1 nmol/mg protein [54].

#### *4.4.2.2. Gold(I) thioglucose*

Gold(I) thioglucose in the presence excess of glutathione (GSH) leads to strong and reversible inhibition of selenium‐GPXs. Gold(I) could competitively combine in reduced form of selenocysteine in active sites, and gold(I) forms a dead‐end complex with glutathione perox‐ idase resulting in suppression of GPXs. So glutathione peroxidase could be a target of gold drugs that used in the treatment of disease caused by excessive activity of GPXs, such as rheumatoid arthritis [55].

#### **4.5. Activators of GPXs**

To our knowledge, most literature studies on enhancing GPXs activity were about how to regulate expression of GPXs, study on the activator by acting the enzyme directly was few and most of them are GPX‐mimetic compounds.

#### *4.5.1. Enhance activity of GPX mimetics*

For some GPX mimetics, its activity can be enhanced by electron‐donating. naphthalene *peri*‐ diselenide mimeticswas increased by electron‐donating methoxy substituents, while a further 100‐fold increase was observed with the corresponding ditelluride. This was attributed to the ability of the methoxy group to stabilize the increasing positive charge at the selenium atom during the rate‐determining step of the catalytic cycle, which involves the oxidation of Se(II) to Se(IV), thus improved their catalytic activity to levels comparable with their aliphatic counterparts [56]. Others report that 6‐bromo‐substituted diselenides also enhanced its activity by threefold [57]. Another strategy is to change the aqueous solubility of the mimetics. Diaryl selenides containing *o*‐hydroxymethylene substituents function as peroxide‐destroying mimetics of the antioxidant selenoenzyme glutathione peroxidase. Several selenide analogues were attached to polyethylene glycol (PEG) oligomers greatly improved aqueous solubility and catalytic activity (10–100 folds) [58].
