**3. CAT**

#### **3.1. The structure and function of CAT**

CAT(EC 1.11.1.6) catalyzes the decomposition of hydrogen peroxide to water and oxygen, widely exists in animals, plants, and microorganisms. Its uniqueness lies in the enzymatic prosthetic group(ferriprotoporphyrin IX) that could catalyze the same reaction as the holoenzyme.

$$
\mathcal{D} \vdash\_2 \bigcirc\_2 \to \bigcirc\_2 + 2 \vdash\_2 \bigcirc \tag{2}
$$

According to the significant catalytic activity, CATs can be divided into three distinct sub‐ groups: typical catalases, a typical catalases and catalase‐peroxidases [23]. Two subgroups, typical catalases and catalase‐peroxidases, contain heme, but the third group has no heme, namely manganese catalases. Most of catalases belong to typical catalases, except catalases in the domain of Archaea. Although there are differences in the primary structure among these typical catalases, but the three‐dimensional structure appears well conserved. Most of these hydroperoxidases are homotetramers with four prosthetic heme groups (**Figure 2(a)**, PDB, 1E93) [24].

**Figure 2.** The three‐dimensional structure of three types of catalases. (a) a typical catalase (depleted in iron) from *Pro‐ teus mirabilis*, (b) a catalase‐peroxidase from *Synechococcus elongatus* PCC7942, (c) a manganese catalase from *Lactobacil‐ lus plantarum.* Color scheme: blue ball—Fe or Mn, red—heme group, others colors indicate different subunit.

Catalase‐peroxidases may originate from ancestral, a relatively large momomeric unit comprising more than 700 amino acids, indicating that they probably from duplication of an ancestral gene [25]. And catalase‐peroxidases show much higher sequence homology with heme peroxidases than with typical catalases. Recently, a three‐dimensional structure of catalase‐peroxidases has been obtained and is shown in **Figure 2(b)** (PDB, 3WXO) [26].

There is little structural information about manganese catalases up to now. Subunits with molecular weights around 30 kDa are recognized as tetramers or hexamers, and are remarkably stable at high temperatures [23, 27]. A crystal structure of a manganese catalase from *Lactoba‐ cillus plantarum* at 1.33a resolution is established (**Figure 2(c)**, PDB, 1O9I) [28].

#### **3.2. Inhibitor of catalase**

#### *3.2.1. Noncompetitive inhibitor*

#### *3.2.1.1. Sodium azide, amine, and cyanide*

Sodium azide (NaN3), amine, and cyanide are nonspecific inhibitors of CAT. Catalase‐ peroxidases are very sensitive to NaN3, a lower concentration of NaN3 could lead to the enzyme lose its activity by 50%. The inhibitory efficiency order was sodium azide>amine>cyanide>3‐ amino‐1,2,4‐triazole(**Table 2**) [29]. A similar result was found in the other typical monofunc‐ tional catalases [30, 31].


**Table 2.** Effect of inhibitors on the catalase and peroxidase activity of the catalase‐peroxidase of *Archaeoglobus fulgidus* [29].

#### *3.2.1.2. 3‐Amino‐1,2,4‐triazole*

HO O HO 22 2 2 2 2 ® + (2)

According to the significant catalytic activity, CATs can be divided into three distinct sub‐ groups: typical catalases, a typical catalases and catalase‐peroxidases [23]. Two subgroups, typical catalases and catalase‐peroxidases, contain heme, but the third group has no heme, namely manganese catalases. Most of catalases belong to typical catalases, except catalases in the domain of Archaea. Although there are differences in the primary structure among these typical catalases, but the three‐dimensional structure appears well conserved. Most of these hydroperoxidases are homotetramers with four prosthetic heme groups (**Figure 2(a)**, PDB,

**Figure 2.** The three‐dimensional structure of three types of catalases. (a) a typical catalase (depleted in iron) from *Pro‐ teus mirabilis*, (b) a catalase‐peroxidase from *Synechococcus elongatus* PCC7942, (c) a manganese catalase from *Lactobacil‐*

Catalase‐peroxidases may originate from ancestral, a relatively large momomeric unit comprising more than 700 amino acids, indicating that they probably from duplication of an ancestral gene [25]. And catalase‐peroxidases show much higher sequence homology with heme peroxidases than with typical catalases. Recently, a three‐dimensional structure of catalase‐peroxidases has been obtained and is shown in **Figure 2(b)** (PDB, 3WXO) [26].

There is little structural information about manganese catalases up to now. Subunits with molecular weights around 30 kDa are recognized as tetramers or hexamers, and are remarkably stable at high temperatures [23, 27]. A crystal structure of a manganese catalase from *Lactoba‐*

Sodium azide (NaN3), amine, and cyanide are nonspecific inhibitors of CAT. Catalase‐ peroxidases are very sensitive to NaN3, a lower concentration of NaN3 could lead to the enzyme

*cillus plantarum* at 1.33a resolution is established (**Figure 2(c)**, PDB, 1O9I) [28].

*lus plantarum.* Color scheme: blue ball—Fe or Mn, red—heme group, others colors indicate different subunit.

1E93) [24].

212 Enzyme Inhibitors and Activators

**3.2. Inhibitor of catalase**

*3.2.1. Noncompetitive inhibitor*

*3.2.1.1. Sodium azide, amine, and cyanide*

3‐Amino‐1,2,4‐triazole (aminotriazole, ATZ) as a noncompetitive catalase‐specific inhibitor is used to study on physiological changes in organisms [32]. Aminotriazole could combine catalase‐H2O2 compound I, thus results in loss of enzymatic activity. In alcohol‐induced liver injury, catalase plays a dual role. On the one hand, catalase could scavenge hydrogen peroxide originated from alcohol to water, but on the other hand, catalase decomposes alcohol that might be harmful to liver, some research studies [33] show that catalase is inhibited by ATZ, which attenuated alcohol‐induced acute liver injury.

#### *3.2.1.3. Salicylic acid*

Salicylic acid acts as an electron donor for the peroxidative cycle of catalase, it is a noncom‐ petitive inhibitor of catalases. It is interesting to note that different CAT salicylic acid exhibits different inhibitory property. CAT1 and CAT2 are two isoenzymes from maize (*Zea mays* L.). The Lineweaver‐Burk plot of SA inhibition of CAT1 and CAT2 shows that CAT1 is noncom‐ petitive manner, while CAT2 is inhibited in a competitive manner [34]. SA has a dual function on catalase, which means SA can both inhibit and activate its activity. Durner and Klessig [35] examined the effects of SA on the formation of the various redox states or reaction intermedi‐ ates of catalase (**Figure 3**). The absorption spectrum of compounds I, II and III was different, thus various redox states or reaction intermediates of catalase can be distinguished spectro‐ scopically by their absorption spectra in the Soret (near UV) region. Through the difference at the absorption spectra of intermediates, SA was confirmed acting as a one‐electron donor that siphons compound I from the extremely fast catalytic cycle into the relatively slow peroxidative cycle (~1000 times slower) by promoting the formation of compound II [36, 37].

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

#### *3.2.2. Competitive inhibitor*

#### *3.2.2.1. Metal ions*

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 that catalase can be inhibited by certain metal ions (including Cu2+, Zn2+, and Ag+ ), a process 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 group and be involved in the enzyme reaction.

#### *3.2.2.2. ρ‐Hydroxybenzoic acid is a competitive inhibitor of catalases from maize*

Phenolic compounds, such as salicylic acid, aspirin, benzoic acid, o‐coumaric acid, and ρ‐ hydroxybenzoic acid play a role in the induction of abiotic stress resistance. But only ρ‐ hydroxybenzoic acid showed the inhibitory effect on two catalases from maize in a competitive manner, the other compounds were in noncompetitive manner. Weak inhibition by ρ‐hydrox‐ ybenzoic acid was also found in both isozymes, only 15 and 9% activity was inhibited, respectively [34].

#### **3.3. Activator of catalases**

Metformin is a commonly used antidiabetic drug with AMP‐activated protein kinase (AMPK)‐ dependent hypoglycemic activities. A recent study [40] shows that metformin can significantly enhance the activity of catalase. Although metformin bound to CAT byinteracting with hydrogen bonds…., metformin did not affect the expression level of catalase, just affecting its activities, such as Lys449, Val450, and Glu455 residues in murine CAT. The preliminary study indicated that metformin might be a new drug to alleviate oxidative injury and enhance the defense ability of antioxidants.
