CATALASE

Michael Joseph D. Dano HUB33 De La Salle University Dasmarias Dasmarias, Cavite, Philippines

INTRODUCTION Catalase is one of the most studied enzymes because of its abundant presence in nature. Catalase is also known as equilase, caperase, optidase, catalase-peroxidase with a systematic name of hydrogen-peroxide oxidoreductase and CAT in abbreviated form. It is an enzyme that is a part of a family of enzymes called peroxidases that usually has a hydrogen peroxide substrate and typically catalyze a reaction of the form: ROOR' + electron donor (2 e-) + 2H+ ROH + R'OH. It is responsible for the catalysis of the breakdown of hydrogen peroxide to water and oxygen. It is found in many organisms that are exposed to oxygen. In mammalian tissues, it prevents and protects the body tissues from the toxic effects done by hydrogen peroxide which is constantly produced by numerous metabolic processes by breaking it down to less reactive gaseous oxygen and water molecules without the production of free radicals. Catalase is one of the most efficient enzymes known with rates approaching 200,000

catalytic events per second per subunit (near the diffusion-controlled limit).

CATALASE: Structure, Function, and Features Catalase Structure Catalase exists as a dumbbell - shaped tetramer of four identical sub units or monomer with each over 500 amino acids long and having a molecular weight of 60,000 u. At its catalytic center, each monomer contains a porphyrin heme prosthetic group which allows it to react to hydrogen peroxide. y Primary Structure o The Catalase monomer is:  composed of more than 500 amino acid polypeptide chain,

  y

one heme group A molecule of NADH.

Secondary Structure o About 60% of Catalase structure is composed of regular secondary structural motifs. It is composed of:    E-helices accounting for 26% of its structure. F-structure for 12%. Irregular structure is composed of predominance of extended single stands and loops that is crucial in the assembly of the tetramer.

Tertiary Structure
o Each monomer has four distinct domains:  The first domain is made up of the amino-terminal 75 residues. These form an arm with two E-helices and a large loop extending from the globular subunit.  The second and largest domain contains the heme moiety. It is composed of residues 76 to 320 and may be classified as a combination ofE+F type domain. It includes a F-barrel, several helical segments of three to four turns each, and various loops. The F-barrel consists of two four stranded anti-parallelF-sheets that twist to form a closed cylindrical surface.  The third domain consists of residues 321-436 and is referred to as the wrapping domain. It lacks discernable secondary structure except for two helices, the largest of which (the essential helix) contains the heme phenol ligand, Tyr357.  The carboxyl-terminal portion of the molecule contains residues 437 to 506 and is folded into a four-helical domain similar to the globin folds. Along with three E-helices from the heme-containing domain, these helices form one surface of the enzyme. Quaternary Structure o Functional Catalase is a tetramer of four identical holo subunits. Each monomer harbors a single heme and NADP. Whereas the NADPs lie on the surface, the heme moieties are embedded in the middle of each monomer, ~20 below the molecular surface, and ~23 from the center of the tetramer.

The assembly of the multimeric complex is presumably more complicated than a simple combination of monomers, with changes in the folding pattern of each monomer occurring so as to optimize packing interactions. Most intersubunit contacts are confined to the amino-terminal arms and the wrapping domains. The most flexible parts of the protein are thus responsible for most of the quaternary structural interactions. The amino-terminal domain becomes almost completely buried between neighboring subunits in the tetramer.F-strands from two pairs of adjacent wrapping domains form intersubunit anti-parallel F-sheets. There are numerous salt bridges at the interfaces between monomers, mostly involving arginine, asparagine, and glutamic acid partners. The tetrameric model shown shows a loss of 10633.2 2 of solvent accessible surface area upon complex formation (D. Marcey, 2001).

Cofactor of Catalase o Heme; Manganese  A protein manganese which containsMn(3+) in the resting state, which also belongs here, is referred to as pseudocatalase. Enzymes from some microorganisms, such as Penicillium simplicissimum, which exhibit both catalase and peroxidase activity, have sometimes been referred to as catalase-peroxidase.

Coenzyme of Catalase o Q10 ( ubiquinone, ubidecarenone)  Is a 1, 4-benzoquinone, Q10 where the letter Q refers to the Quinone chemical group, and the number 10 refers to the number of isoprenyl chemical subunits. This oil-soluble chemical is present primarily in the mitochondria of eukaryotic cells. It is a component of the electron transport chain and participates in aerobic cellular respiration, and is crucial in the production of ATP. 95% percent of the human bodys ATP is generated this way. Therefore, those organs with the highest ATP requirementssuch as the heart, liver and kidney mostly major organs, have the highest Coenzyme Q10 concentrations.

Figure 1.0 PDB rendering of Catalase based on 1dgb.

Figure 2.0 the Active Site of Catalase/ Heme Cavity (Prieto et al., 2009)

Figure 3.1 Heme; Manganese Structure Cofactor of Catalase

Figure 3.2 Q10 Coenzyme of Catalase

Mechanism of Catalysis The mechanism of catalase catalysis has not been fully known yet, but the catalysis is thought to occur in two stages similar to that of cytochrome c peroxidases: H2O2 + Fe (III)-E H2O + O=Fe (IV)-E (. +) H2O + Fe (III)-E + O2

H2O2 + O=Fe (IV)-E (. +)

In this reaction, Fe-E represents the Iron (Fe) center of the heme attaching itself to the rest of the enzyme (E).

As Hydrogen peroxide enters the heme cavity (active site), it will be severely sterically 74 147. hindered therefore it must interact with His and Asp It is in this position where the first step of catalysis occurs. A proton (hydrogen ion) then is transferred between oxygen atoms of 74 the peroxide via His . This then causes the O-O bond to elongate and polarize which then breaks heterolytically as hydrogen peroxide is then coordinated to the iron center. This results to the displacement of water and then forms Fe (IV) =O and a heme radical. The radical degrades in an electron transfer to rid of the radical electron leaving the heme ring unaltered. For the

second stage, in a similar two electron transfer reaction, Fe (IV)=O reacts with a second hydrogen peroxide to form the original Fe (III) E, another H2O and another molecular mole of oxygen.
357 The reactivity of the heme (iron center) is enhanced by phenolate ligand of Tyr in the

fifth iron ligand which helps in the oxidation of Fe (III) and Fe (IV) and the removal of an electron 74 in the heme ring. The efficiency of catalase may be caused by the interaction of His and 147 Asp with interaction intermediates. The mechanism is supported by an experiment 74 supporting evidence that modification of His with 3-amino-1, 2, 4-triazole inhibits the enzyme by hindering substrate binding. The rate of the reaction can be determined by the Michaelis Menten equation. Catalase can also oxidize different toxins such as formaldehyde, phormic acids, phenols and alcohols. With it, it uses hydrogen peroxide according to the following reaction: H2O2 + H2R 2H2O + R

Again, the exact mechanism is unknown.

Figure 3.0 Mechanism of Catalysis of Catalase. (Regelsberger et al., 2001)

Kinetics of Reaction Hydrogen peroxide is broken down slowly in aqueous solutions but is accelerated by many types of catalysts more specifically peroxidases. The reaction is typically first order in catalyst concentration and H2O2. With enzyme catalase the reaction is zero order in H2O2 (rate depends only in amount of catalyst). At low concentrations, Michaelis-Menten kinetics predicts that the rate will become first order for the substrate (peroxide). The reaction rate of the enymatic decomposition of hydrogen peroxide increases in the available temperature range with rising temperature. A linear relationship exists between the catalase concentration and the reaction rate. Possible inhibitors for catalase are cyanide, phenols, azide and urea. All these inhibitors are competitive inhibitors, and connect to the catalase directly, thus reducing drastically the catalase's potential to exercising its catalytic action. Another possible inhibitor is hydrogen peroxide in high concentrations, which poisons the enzyme system. Up to 0.4M H2O2 concentration, the rate is proportional to the concentration of the substrate (P. Keusch, 2003). At higher concentrations of H2O2, the rate of the reaction is dropped off. Also, any heavy metal ion such as copper cations in copper (II) sulfate may play a non competitive inhibitor role. An activator is any molecule that is when it binds to an enzyme increases its activity. (Meronba?!)

Mode of Regulation An examination of three inbred strains of mice differing with respect to liver and kidney catalase activity reveals two distinct genetic factors controlling the level of liver catalase activity. The first genetic factor controls the catalytic activity of the enzyme. Specific activity of purified enzyme from C57BL/6 and C57BL/Ha strains is 60% of that of the DBA/2 strain. The second factor controls the content of liver catalase. Liver catalase of C57BL/Ha is degraded in vivo at a rate one half that of liver catalase of DBA/2 and C57BL/6, resulting in the accumulation of twice as many catalase molecules in C57BL/Ha. The factor affecting turnover of catalase is apparently specific for catalase of liver since no differences exist in kidney catalase levels between C57BL/Ha and C57BL/6. Furthermore, this factor does not appear to alter the metabolism of total liver protein since no substantial difference in the turnover rate of liver protein is observed among the strains. It is particularly significant that the genetic factor affecting the amount of liver catalase does so by altering the rate of catalase degradation rather than the rate of synthesis, confirming the previously published report of Rechcigl and Heston (1967). Thus, these studies emphasize that the quantity of an enzyme in animal cells is a

balance between the rate of synthesis and the rate of degradation of the enzyme (Ganschow, 1970). Application and Associated Disease Application o In food processing, catalase is used to remove hydrogen peroxide from milk before manufacturing it into cheese. o o In food wrappers, it prevents the food being wrapped from oxidizing. In the textile industry, catalase is used in fabrics to make it free from hydrogen peroxide. o In contact lens cleaning, some contact lens cleaning products uses hydrogen peroxide to disinfect the lenses, a solution then containing catalase is used to decompose the hydrogen peroxide before using the lens once more. o Its use in Microbiology is that the catalase test is done to identify species of bacteria. The test is done by placing a drop of hydrogen peroxide on a specimen slide. Using an applicator stick, a scientist smears the sample colony into the hydrogen peroxide drop.    If bubbles or froth forms, the organism is said to be catalasepositive. Staphylococci and Micrococci are catalase-positive. If not, the microorganism is said to be catalase-negative. Streptococci and Enterococci are catalase-negative. The catalase test by itself cannot pinpoint a specific organism, combined with other tests such as antibiotic resistance; it remains very helphul in diagnosis. The presence of catalase in bacterial cells depends on both the growth condition and the medium used to grow the cells. y Associated Diseases o Acatalasia is a genetic (autosomal recessive) disorder caused by the complete lack of catalase. This disease is relatively benign and asymptomatic; this may cause oral ulcerations and gangrene.