BB 404 Supplement/1999.
OXIDATIVE STRESS
INCLUDING GLUTATHIONE, A PEPTIDE FOR CELLULAR DEFENSE AGAINST OXIDATIVE STRESS.
JAMES A. THOMAS
DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS
IOWA STATE UNIVERSITY
AMES, IA. 50011
Tel 515-294-3434
Fax 515-294-0453
Email jat@iastate.edu
INTRODUCTION
RADICALS AND NON-RADICALS IN OXIDATIVE STRESS
A. Oxygen species
B. Nitrogen species
EFFECTS OF OXIDANTS ON MACROMOLECULES
A. Carbohydrates
B. Nucleic Acids
C. Proteins
D. Lipids
CELLULAR ANTI-OXIDANTS
A. The Glutathione Redox Cycle and the Protein S-thiolation Cycle.
B. Vitamin E and Membrane Peroxidation
C. Enzymes as Anti-oxidants in cells
D. Other Uses of Glutathione that require different chemistry.
This addition to the text material acts as a supplement to the lecture material on glutathione and oxidative stress.
Off-Campus students: Some of the material is not found on the videotapes, but you are still responsible for this material on exams.
INTRODUCTION
Let's begin with a description of the problem that cells encounter by using oxygen. Oxygen is the primary oxidant in metabolic reactions designed to obtain energy from the oxidation of a variety of organic molecules. Oxidative stress results from the metabolic reactions that use oxygen, and it has been defined as a disturbance in the equilibrium status of pro-oxidant/anti-oxidant systems in intact cells. This definition of oxidative stress implies that cells have intact pro-oxidant/anti-oxidant systems that continuously generate and detoxify oxidants during normal aerobic metabolism. When additional oxidative events occur, the pro-oxidant systems outbalance the anti-oxidant, potentially producing oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately leading to cell death in severe oxidative stress. Mild, chronic oxidative stress may alter the anti-oxidant systems by inducing or repressing proteins that participate in these systems, and by depleting cellular stores of anti-oxidant materials such as glutathione and vitamin E.
A disturbance in pro-oxidant/anti-oxidant systems results from a miriad of different oxidative challenges, including radiation, metabolism of environmental pollutants and administered drugs (these are xenobiotics, i.e., foreign materials), and immune system response to disease or infection. The immune response is especially interesting since many toxic oxidative materials are generated in order to kill invading organisms. Evidence for the role of a variety of chemicals called radicals in these processes has led to interest in the reactions of partially reduced oxygen products and radical and non-radical species derived from them. A variety of reactive nitrogen species derived from the reactions of nitric oxide play important roles as well. A radical species is specifically understood to be any atom that contains one or more orbital electrons with unpaired spin states. The radical may be a small gas molecule such as oxygen or nitric oxide, or it may be a part of a large biomolecule such as a protein, carbohydrate, lipid, or nucleic acid. Some radical species are very reactive with other biomolecules and others like the normal triplet state of molecular oxygen are relatively inert.
Oxidative stress has been implicated in human disease by a growing body of facts. However, cells have multiple protective mechanisms against oxidative stress and succeed in preventing cell damage to the extent that these protective mechanisms are effective. Many dietary constituents are important sources of protective agents that range from anti-oxidant vitamins and minerals to food additives that might enhance the action of natural anti-oxidants. Indeed, at least part of the beneficial effects of a high fruit and vegetable diet is thought to derive from the variety of plant anti-oxidants that might act as beneficial supplements in humans. On the other hand, materials such as pesticides, polyunsaturated lipids, and a variety of plant and microorganism-derived toxins might produce pro-oxidant effects in man. This chapter deals with our current understanding of the molecules that cause oxidative stress at the cellular level, with the anti-oxidant systems that function at the cellular and organismal level, and with the role of dietary materials in oxidative stress and human disease.
Glutathione is a peptide found in most every cell at a high concentration (see figure 8 for its structure). Its unique features include a) high water solubility, permitting cells to use high concentrations of the peptide, b) an amino acid constituent (cysteine) that is readily oxidized and reduced under the mild conditions required in cell metabolism, and c) an unusual peptide bond that prevents nonspecific destruction by hydrolytic enzymes that attack normal peptide bonds. The chemistry and biology of this important peptide provide an excellent example to begin study of peptide and protein chemistry. This chapter provides more information on the biological role of glutathione, and some of its unique chemical properties on page 6.
RADICAL AND NON-RADICALS IN OXIDATIVE STRESS
A. OXYGEN SPECIES
Radicals of oxygen (superoxide anion, hydroxyl radical, and peroxy radicals), reactive non-radical oxygen species such as hydrogen peroxide and singlet oxygen, as well as carbon, nitrogen, and sulfur radicals comprise the variety of reactive molecules that can constitute an oxidative stress to cells. It has been estimated that a maximum of 5 % of the total oxygen metabolism of liver tissue results in the production of partially reduced oxygen species such as those shown. This represents a significant stress under normal conditions, and there is evidence that some cellular damage occurs under these conditions.
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Oxygen O2 |
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ß ------ ---Arrows ----- the addition of one electron to the molecule above. |
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Superoxide Anion · O2- |
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ß |
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Hydrogen Peroxide H2O2 |
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ß |
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Hydroxyl Radical · OH |
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ß |
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Water H2O |
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Atmospheric oxygen, although a radical, is not particularly reactive with biological molecules because the two orbital electrons participating in oxidation reactions have the same spin state. Thus, electrons that might be added to these orbitals during reduction of oxygen must be added singly rather that as a pair of electrons with paired spins. This spin restriction prevents rapid reactions with compounds that could easily react without the spin restriction. Any process that produces a one electron reduction of oxygen produces the more reactive radical called superoxide anion. A second form of oxygen, i.e., singlet oxygen, is a much more reactive form with paired electrons. Reduction of this form of oxygen does not have the same spin state restriction.
Superoxide anion (O2
·- ) is generated continuously by several cellular processes including the microsomal and mitochondrial electron transport systems. In addition, xanthine dehydrogenase/oxidase and other cellular oxidases may be important sources of this molecule. Cells of the immune system have a special role in production of superoxide anion since they contain a membrane-bound enzyme complex, the NADPH oxidase, that reduces oxygen with NADPH to produce copious amounts of superoxide anion. The products of this reaction are essential for effective bacterial killing. Absence of this enzyme activity is responsible for an inherited human condition called chronic granulomatous disease that is characterized by recurrent infections. The universal presence of superoxide dismutase (an enzyme described on page 7) in both cytoplasm and mitochondria insures that much of the superoxide anion is rapidly converted into hydrogen peroxide. Superoxide anion is not a particularly reactive molecule and it can diffuse considerable distances from its site of production. It may combine with other reactive species such as nitric oxide to yield a more reactive species, peroxynitrite (Next section).Hydrogen peroxide (H2O2) is a non-radical molecule (paired electrons) generated by the same sources that produce superoxide anion since two molecules of superoxide anion dismute to hydrogen peroxide and oxygen readily. Superoxide dismutase catalyzes this reaction. There are also a number of other specific enzymes that produce hydrogen peroxide directly. Hydrogen peroxide can diffuse over considerable distances and may pass membranes readily in this process. Thus, pools of hydrogen peroxide equilibrate rapidly. Hydrogen peroxide and superoxide anion can occur both inside and outside cells.
In the presence of a transition cation such as iron or copper, superoxide anion can give rise to the highly reactive hydroxyl radical species (HO·) by the Haber-Weiss reaction (see below).
HABER-WEISS REACTION
O2·- + H2O2 Þ O2 + HO- + HO·
Iron catalyzes the reaction by the following mechanism. Summing the 2 reactions together gives the reaction above.
FeIII + O2
·- Þ FeII + O2FeII + H2O2
Þ FeIII + HO- + HO·
Hydroxyl radical is considered to be a principal actor in the toxicity of partially reduced oxygen species since it is very reactive with all kinds of biological macromolecules, producing products that cannot be regenerated by cell metabolism (Figure 2 illustrates). The rate of reaction of hydroxyl radical is diffusion-controlled and it reacts very close to its site of production. Therefore, damage by this radical is very site-specific.
FIGURE 2. Reaction of Hydroxyl Radicals with Biological Molecules.
Peroxyl radicals occur during the oxidation of lipids or other organic molecules in oxidative stress. They are formed by addition of oxygen to alkyl radicals (carbon radicals- REACTION 1 below). The peroxyl radical species, which are not very reactive, may diffuse a considerable distance. They have been shown to react avidly with sulfhydryl groups (thiols) to generate the thiyl radical (REACTION 2 below).
Singlet oxygen is formed by oxidation of other partially reduced oxygen species, resulting in an oxygen with paired elections in the reactive orbital.
The variety of oxygen species described above indicate the complexity of the reactions that can result from an oxidative stress. Factors such as the site of production, the availability of transition metals, and the action of enzymes determine the fate of each radical species and its availability for reaction with cellular molecules. The H2O2 concentration under steady state conditions in liver has been estimated to be 10-7 - 10-9 M, while superoxide anion is 10-11 M.
B. NITROGEN SPECIES
Nitric oxide (NO
·) is an abundant reactive radical that acts as an important oxidative biological signal in a large variety of diverse physiological processes, including smooth muscle relaxation, neurotransmission, and immune regulation. The nitric oxide synthase enzyme (Figure 3a) generates this nitrogen based radical species by a five electron oxidative reaction that utilizes arginine as its substrate. This enzyme occurs in several different regulated forms in different cell types and it is highly regulated by Ca++ and other factors. Cells of the immune system produce both superoxide anion and nitric oxide during the oxidative burst triggered during inflammatory processes. Under these conditions, nitric oxide and superoxide anion may react together to produce significant amounts of a much more oxidatively active molecule, peroxynitrite (Figure 3b). Peroxynitrite is a strong oxidant that attacks protein cysteines and methionines. It also reacts with protein tyrosines by adding an NO2 to the ring of these amino acids. The protein modifications that result from peroxynitrite production may provide explanations for some of the observed biological effects of NO·. Additionally, NO· adds to thiols to form S-nitrosothiols (Figure 3b). The sulfhydryl is formally oxidized by one electron in this reaction and subsequent reaction with a reduced thiol leads to the formation of a disulfide. S-nitrosothiols may provide a mechanism for transport of NO· in the blood plasma where the low abundance of reduced thiol compounds prevents further reaction of the NO·/thiol adduct.
FIGURE 3.
Formation and Reactions of Nitric Oxide. A) The reaction catalyzed by nitric oxide synthase. The exact stoichiometry of the reaction is not known. The five electron reaction generates nitric oxide and oxidizes NADPH in the process. B) Two of the important by-products of nitric oxide generation are shown. Peroxynitrite is a simple addition product in which no oxidation or reduction is necessary. S-nitrosothiols are one electron oxidation products of the reaction between thiols and nitric oxide. The reaction requires some electron accepting species.
EFFECTS OF OXIDANTS ON MACROMOLECULES
A. CARBOHYDRATES
Hydroxyl radicals react with carbohydrates by randomly abstracting a hydrogen atom from one of the carbon atoms, producing a carbon- centered radical. This leads to chain breaks in important molecules like hyaluronic acid (Figure 4) in a process involving intermediates such as peroxyl radicals. In the synovial fluid surrounding joints, an accumulation and activation of neutrophils during inflammation produces significant amounts of oxyradicals. This phenomenon apparently accounts for a significant decrease in the synovial fluid of affected joints.
FIGURE 4. Reaction of Hydroxyl Radicals with Polysaccharides (Hyaluronic Acid).
B. NUCLEIC ACIDS
Nucleic acids are pentose-phosphate polymers that can undergo reactions with hydroxyl radical like those depicted for hyaluronic acid (Figure 4). In addition there are several important examples of modifications to the base portion of the polymer (Figure 5). In fact these base modifications may be responsible for genetic defects produced by oxidative stress. Recently, 8-hydroxy guanosine has generated considerable interest as a product of hydroxyl radical attack on DNA that can be used to estimate DNA damage in humans. In humans oxidative damage to DNA has been estimated as 104 hits per cell per day. Estimation of modified bases in urine is a useful means of assessing the amount of DNA damage in an animal. Products such as 8-hydroxy guanosine, thymidine glycol, and uric acid are used for these estimates. DNA damage has also been estimated by chain breaks and base modifications in cultured cells under oxidative stress. An important metabolic effect of DNA damage is the rapid induction of polyadenosine diphosphate ribose synthesis (ADP-ribosylation) in nuclei, resulting in extensive depletion of cellular NADH pools. ADP-ribosylation has been associated with repair of damaged DNA.
C. PROTEINS
Proteins have many reactive sites that can be damaged during oxidative stress, but interest has centered on three measurable events. First, aggressive radicals such as hydroxyl radical can fragment proteins in plasma, and the fragmented products of specific proteins, if known, can be detected. This fragmenting is associated with reactions at specific amino acids such as proline (Figure 6a) and histidine. Second, proteins may contain metal binding sites that are especially susceptible to oxidative events through interaction with the metals. These reactions usually produce irreversible modifications in amino acids that might be involved in metal ion binding, e.g., histidine. These modifications may produce signal sequences that are recognized by specific cellular proteases that degrade such proteins. Finally, many intracellular proteins have "reactive" sulfhydryls groups on specific cysteine residues (See ANTI-OXIDANTS) that can be modified (oxidized) to specific forms (disulfides) that can be reduced again by metabolic processes. Similarly, some proteins have a "reactive" methionine that can undergo a reversible modification to methionine sulfoxide (Figure 6b). The disulfide and sulfoxide forms of these two amino acids may actually serve a protective role, since the metabolic reversibility of the protein modification effectually detoxifies the oxidative species that caused the modification. The reversible nature of the modifications of cysteine and methionine also suggests that oxidative modifications of this type may have a role in regulating metabolic events in the cells under oxidative stress.
FIGURE 6. Methionine and Proline Oxidation. A) The reaction shows the irreversible oxidation of proline in a peptide. The result is a break in the polypeptide chain and the introduction of new carboxyl groups that can be measured to quantitate these events. B) The reactions demonstrates the reversible oxidation/reduction of methionine as it occurs in several proteins. The reduction is an enzymatic process that requires a reduced thiol such as glutathione
D. LIPIDS
Lipid peroxidation of polyunsaturated lipids is a facile process. This oxidation affects materials that are prevalent in dietary constituents, seriously affecting the flavor of foods. In intact cells these materials are major constituents of cellular membranes, where peroxidation of membrane lipid seriously impairs membrane function. Most peroxidized membrane lipid occurs as a result of oxidative stress in intact cells, but some dietary material may be directly incorporated into cell structures. Lipid peroxidation is a radical-initiated chain reaction that is self-propagating in cellular membranes. As a result, isolated oxidative events may have profound effects on membrane function. The reactions of this process are depicted in Figure 7.
FIGURE 7. Reactions of Lipid Peroxidation.
The products of lipid peroxidation are easily detected in blood plasma and have been used as a measure of oxidative stress. The most commonly measured product is malondialdehyde (Figure 7). In addition the unsaturated aldehydes produced from these reactions have been implicated in modification of cellular proteins and other materials. The peroxidized lipid can produce peroxy radicals and singlet oxygen by reactions discussed above. Vitamin E is particularly effective as an anti-oxidant in biological membranes and in lipid particles found in blood plasma.
CELLULAR ANTI-OXIDANTS
The most effective anti-oxidant in oxidative stress is dependent on the specific molecules causing the stress, i.e., superoxide anion, lipid peroxides, iron-generated hydroxyl radical, etc., and the cellular or extracellular location of the source of these molecules. As an example, damage to a cell membrane occurs from both internally and externally generated oxidative stress. This damage is most effectively prevented by vitamin E which reacts with peroxyl and hydroxyl radicals, carotenoids which react with singlet oxygen, and possibly by membrane bound proteins. The chain-breaking anti-oxidant function of vitamin E in membranes results from its close association with polyunsaturated components of the membrane. It can be regenerated by reaction with cytoplasmic vitamin C and glutathione, or by membrane-bound quinols. Vitamin C is subsequently reduced by glutathione through the glutathione cycle that is described below. Thus, a specific attack on membranes results in the participation of at least three different anti-oxidants. Similarly, when oxidative stress occurs in plasma a variety of different anti-oxidants participate in the response. Many plasma proteins are affected by the process, causing either irreversible or reversible loss of functional protein activity. A good example is the oxidation of methionine residues in alpha 1-protease inhibitor (an inhibitor of elastase). The modification can be reversed by a specific reductase enzyme that restores that activity of the inhibitor (see Figure 6).
A. THE GLUTATHIONE REDOX CYCLE AND THE PROTEIN S-THIOLATION CYCLE.
The low molecular weight thiol, glutathione, and "reactive" protein sulfhydryls (exposed cysteines in many proteins) are primary participants in cellular anti-oxidant systems. Glutathione (Figure 8) is abundant (3 to 10 mM) in cytoplasm, nuclei, and mitochondria and is the major soluble anti-oxidant in these cell compartments. Reactive protein sulfhydryls are abundant in both soluble proteins and in membrane-bound proteins.
FIGURE 8. Structure of Glutathione.
The sulfur atom in sulfhydryl groups easily accommodates the loss of a single electron (reaction 1 below) and the lifetime of radical species of sulfur, i.e., a thiyl radical, may be significantly longer than many other radicals generated during stress. Sulfhydryl groups also partially ionize at cellular pH values producing the more reactive nucleophile, thiolate anion (reaction 2). The pKa of the sulfhydryl group of glutathione is 9.3 and many other sulfhydryl groups, especially those on proteins, may have considerably lower pKa’s as a result of local electronic effects on the functional group. The thiolate anion is responsible for the reactivity of thiols with a variety of foreign materials in conjugation reactions during xenobiotic metabolism (see the last section of this chapter). Thus, the reactions of sulfhydryl groups during oxidative stress include examples in which both sulfur radicals and thiolate anions are important.
(1) one electron oxidation: O2·- + H+ + Glutathione-SH
Þ glutathione-S· + H2O2or O2
·- + H+ + Protein -SH Þ protein -S· + H2O2(2) ionization: (no oxidation involved) glutathione-SH
Þ glutathione-S - + H+Protein -SH
Þ protein -S - + H+The enzymes of the glutathione redox cycle and the protein S-thiolation cycle are shown in Figure 9a. The glutathione redox cycle is primarily mediated by enzyme catalyzed reactions. Glutathione is oxidized by hydrogen peroxide to glutathione disulfide by the selenium-containing enzyme, glutathione peroxidase, and also by other enzymes that may use lipid peroxides rather than hydrogen peroxide as the oxidant. Thus, glutathione can detoxify both soluble and lipid peroxides. Glutathione disulfide is subsequently reduced by glutathione reductase, using NADPH as the reductant. Cellular NADPH, produced by the pentose-P pathway and other cytoplasmic sources, provides the major source of reducing power for detoxifying many peroxides.
The concentration of cellular glutathione has a major effect on its anti-oxidant function and it varies considerably as a result of nutrient limitation, exercise, and oxidative stress. Under oxidative conditions, the concentration of glutathione can be considerably diminished through conjugation to xenobiotics, and by secretion of both the glutathione conjugates and glutathione disulfide from the affected cells. A considerable amount of glutathione may also become protein-bound during severe oxidative stress. Recently, compounds that can both increase and decrease the glutathione concentration when administered to animals have been developed. Some of these compounds may provide the means to modify glutathione concentration in humans in the future. Experiments have already shown beneficial effects in autoimmune disease where glutathione concentrations are reduced.
FIGURE 9.
Redox Cycles of Cellular ThiolsA) The figure on the left shows the glutathione and protein S-thiolation redox cycles. Glutathione is oxidized to glutathione disulfide primarily by hydrogen peroxide and the enzyme glutathione peroxidase. It is reduced again by the enzyme, glutathione reductase, that requires NAPDH. These two enzymes are discussed in more detail in the text (section C, below). Proteins are oxidized to S-thiolated forms by the direct action of several oxidants without enzyme catalysis. S-thiolated proteins are reduced again by the protein, glutaredoxin.
B) Reduction of S-thiolated proteins by glutaredoxin requires several steps of a reactions named thiol-disulfide exchange. Glutaredoxin contains a dithiol active site that is uniquely suited for this purpose because the protein has a binding site for glutathione to position that molecule next to the appropriate thiol in the active site. The mechanism shows that glutathione reduces glutaredoxin which then reduces (dethiolates) the S-glutathiolated protein.
The protein S-thiolation cycle (Figure 9a) shows the effects of reactive oxidizing molecules on proteins that contain at least one reactive sulfhydryl. During oxidative stress, a large family of proteins that contain reactive sulfhydryls are modified by oxidation to mixed-disulfides with attached glutathione (S- thiolation). Other oxidized forms of protein sulfhydryls may occur in special circumstances. One such case may be when the glutathione concentration is not sufficient to react with the protein sulfhydryl and irreversible oxidation to the sulfonic acid occurs by reaction with molecular oxygen. Another case might be found in proteins where adjacent protein sulfhydryls can react together to form a protein disulfide. The potential to form protein disulfides is not great for intracellular proteins, although these modifications are abundant in extracellular proteins. Figure 9a illustrates the protein oxidation cycle that involves formation of S-thiolated proteins. In Figure 9b the mechanism for the reduction of S-thiolated proteins by the low molecular weight protein, glutaredoxin is illustrated. Glutaredoxin is kept reduced by the glutathione pool. It binds to and reduces proteins that have glutathione attached by S-thiolation, by virtue of the specific binding site for glutathione at the active site of the protein. This protein is uniquely designed to reduce S-thiolated proteins.
B. VITAMIN E AND MEMBRANE PEROXIDATION
Vitamin E refers to a family of related compounds (tocopherols) that have polar hydroxylated aromatic rings (chromanol rings) and non-polar isoprenoid side chains. The molecule is lipophillic and resides almost exclusively in cell membranes where the chromanol ring may be at the surface of the membrane and the isoprenoid chain inserted into the non-polar bilayer. Since lipid peroxidation occurs on unsaturated fatty acid chains that reside within the lipid bilayer, and the chromanol ring is the active radical quenching part of the vitamin, the function of vitamin E as an anti-oxidant must involve considerable movement of the lipids and vitamin E to promote molecular interaction. The reactions in which a chromanol ring can participate in these processes are shown in the Figure 10. This figure shows that vitamin E is a chain-reaction breaking anti-oxidant since it quenches the intermediate in the chain reaction. The ascorbate radical formed in this process reacts rapidly with the reduced glutathione pool or with a specific vitamin C reductase enzyme.
FIGURE 10.
Oxidation of Vitamin E By Lipid Radicals and Reduction by Vitamin C.C. ENZYMES AS ANTI-OXIDANTS and DETOXICANTS IN CELLS
Superoxide dismutase (SOD) is a very important enzyme that functions as a cellular anti-oxidant. It is present in cell cytoplasm (copper-zinc enzyme) and in mitochondria (manganese enzyme) in order to maintain a low concentration of superoxide anion. It catalyzes the dismutation of superoxide anion in the following manner.
2 O2
l- + 2 H+ Þ O2 + H2O2The absence of this enzyme is lethal. The amount of superoxide dismutase is controlled by specific redox-sensitive genes in cells. There is also an extracellular form of superoxide dismutase in plasma, lymph, and synovial fluid that is different from the intracellular forms of the enzyme . The extracellular enzyme may function at cell surfaces.
Catalase is a heme protein that catalyzes the reaction shown below in which hydrogen peroxide is detoxified. It is usually found in peroxisomes except in cells like erythrocytes that do not contain these organelles. In that case catalase is a cytoplasmic enzyme. Catalase provides a protective role that is similar to that of glutathione peroxidase because both are important means of removing hydrogen peroxide. Both catalase and glutathione peroxidase are important in hydrogen peroxide detoxification.
2 H2O2
Þ O2 + 2 H2O
Glutathione peroxidase is a cytoplasmic and mitochondrial enzyme that is important for detoxifying H2O2 in most cells. This protein is a seleno protein, i.e., it contains a selenocysteine amino acid at the active site instead of a normal cysteine. The selenium that replaces the normal sulfur in this amino acid has enhanced nucleophillic properties and ionizes more readily to release a proton. It is a much more effective catalyst in the reaction catalyzed by this enzyme.
The flavoprotein, glutathione reductase (the enzyme uses bound FAD, flavin adenine dinucleotide, in an interesting electron transfer reaction), uses the reducing power for the pentose phosphate pathway (NADPH) to keep the glutathione pool in cell in a very reduced state. Even when large amounts of hydrogen peroxide are present this enzyme is very effective at reducing the cellular glutathione pool. Cells contain at least 100 reduced glutathione molecules for every molecule of glutathione disulfide
The net result of this cycle is to use NADPH to reduce hydrogen peroxide to water, a process that requires two electrons. Other reductases can also catalyze reactions that reduce lipid peroxides, i.e., LipidOOH, instead of hydrogen peroxide. Thus the reaction is;
There are a number of other enzymes that contribute to less well defined antioxidant functions of specific cells. These enzymes are usually quite abundant and the amounts present usually reflect the oxidative status of the cell.
D. OTHER USES OF GLUTATHIONE THAT REQUIRE DIFFERENT CHEMISTRY.
The sulfur atom in cysteine is able to partially ionize at neutral pH, i.e., the pH found in normal cells. This produces a different reactivity than the chemistry associated with a sulfhydryl group. Therefore in the case of glutathione there are two forms that differ only in the presence or absence of the proton on the cysteine moiety of the peptide.
glutathione-SH Þ glutathione-S - + H+
The anionic form of glutathione is a strong nucleophile. Since there is very little of the anion at pH = 7 (remember the pK of this ionization is 9.3), cells have developed a family of enzymes called glutathione transferases that make glutathione a more reactive nucleophile by 1) binding glutathione in such a way that the sulfur is induced to ionize more completely, and 2) binding a second molecule close by so that a reaction can be facilitated. This reaction is necessary to detoxify xenobiotic materials, i.e., toxins, drugs, and other foreign compounds. A good example of this reaction is a reaction where glutathione sulfur (a nucleophile) attacks and adds to the electron deficient carbon of the epoxide on the leukotriene forming a new covalent bond between the two molecules (see the left-hand figure on the next page). In many cases an "epoxide" of similar electrophillic center is generated in a foreign compound by a specific enzyme-catalyzed oxidation using a cytochrome-containing oxidase. This attached glutathione now provides a "handle" that can be recognized by other enzymes needed to metabolize compounds that are not normally recognizable. For example, a membrane transport protein may use the "handle" to secrete the offending molecule.
You should also be aware of the fact that glutathione is synthesized by specific enzymes that utilize ATP for its synthesis. This metabolic pathway is very important because the various uses of glutathione constantly deplete the cellular pool of this material and continued synthesis is required. In fact, during periods of strong oxidative stress, this pathway is rapidly activated to provide the needed glutathione. Each reaction of the biosynthesis uses one molecule of ATP to drive the synthesis of a specific peptide bond and the enzyme that catalyzes each reaction provides the specific chemistry needed to bring three molecules together for that reaction.
References