Metalloenzyme; Antioxidant & Their Relationship With Aging, Cancer &Metabolic DisordersMs. Latika Yadav (Research Scholar), Dept. of Foods and Nutrition, College of H.Sc,Maharana Pratap University of Agriculture and Technology, MPUAT, Udaipur, rajasthan-313001, email.id: firstname.lastname@example.org
Metal plays roles in approximately one-third of the known enzymes. Metals may bea co-factor or they may be incorporatedinto the molecule, and these are known asmetalloenzymes.A metalloenzyme is an enzymatic proteinthat has a strong link between its proteinpart and metal , where the metal isembedded with in the molecule. In this casemetal ion is bound tightly to the enzymeand is not dissociated even after severalextensive steps of purification.
Metal plays a variety of roles such as :•They help in either maintaining or producing( orboth), active structural conformation of theenzyme,•Formation of enzyme substrate complex•Making structural changes in substrate molecule,•Accept or donate electrons,•Activating or functioning as nucleophiles, and•Formation of ternary complexes with enzymes orsubstrate.
Physiological Roles of Metal Ions• cell replication• energy production (ATPsynthesis)• O2 transport and storage• synthesis of neurotransmitters• counteracting the effects of aging• RNA synthesis• alcohol breakdown in your liver• hormone synthesis• dilating blood vessels• photosynthesis
It is estimated that approximately half of all proteinscontain a metal. In another estimate, about one quarterto one third of all proteins are proposed require metalsto carry out their functions.Thus, metalloproteins havemany different functions in cells, such as•enzymes, transport and storage proteins, and• signal transduction proteins.• 33% of all enzymes contain transition metal ions!• Metal ions PROMOTE REACTIONS• bond cleavage• bond formation• electron transfer• atom transfer• Metal ions PROMOTE PROTEIN FOLDING
Coordination chemistry principlesIn metalloproteins, metal ions are usually coordinated bynitrogen, oxygen or sulfur centres belonging to amino acid residues of the protein.These donor groups are often provided by side-chains onthe amino acid residues. Especially important are theimidazole substituent in histidine residues, thiolatesubstituents in cysteinyl residues, and carboxylate groupsprovided by aspartate.In addition to donor groups that are provided by aminoacid residues, a large number of organic cofactorsfunction as ligands. Perhaps most famous are thetetradentateN4 macrocyclic ligands incorporated into the hemeprotein. Inorganic ligands such as sulfide and oxide arealso common.
Storage and transport metalloproteinsOxygen carriersHemoglobin, which is the principal oxygen carrier inhumans has four sub-units in which the iron(II) ion iscoordinated by the planar, macrocyclic ligandprotoporphyrin IX (PIX) and the imidazole nitrogenatom of a histidine residue. The sixth coordination sitecontains a water molecule or a dioxygen molecule.myoglobin has only one such unit. The active site is located in anhydrophobic pocket. This is important as, without it,the iron(II) would be irreversibly oxidised to iron(III). Haemoglobin The equilibrium constant for theformation of HbO2 is such that oxygen is taken up orreleased depending on the partial pressure of oxygenin the lungs or in muscle. In hemoglobin the four sub-units show a cooperativity effect which allows foreasy oxygen transfer from hemoglobin to myoglobin.
Hemerythrin is another iron-containing oxygen carrier. The oxygenbinding site is a binuclear iron center. The iron atoms arecoordinated to the protein through the carboxylate side chains of aglutamate and aspartate and five histidine residues. The uptake ofO2 by hemerythrin is accompanied by two-electron oxidation ofthe reduced binuclear center to produce bound peroxide (OOH-).Hemocyanins carry oxygen in the blood of most molluscs, andsome arthropods such as the horseshoe crab. They are second onlyto hemoglobin in biological popularity of use in oxygen transport.On oxygenation the two copper(I) atoms at the active site areoxidised to copper(II) and the dioxygen molecules is reduced toperoxide, O22-.
CytochromesIron(II), can easily be oxidized to iron(III). This functionality isused in cytochromes which function as electron-transfer vectors.The presence of the metal ion allows metalloenzymes toperform functions such as redox reactions that cannot easily beperformed by the limited set of functional groups found inamino acids. The iron atom in most cytochromes is contained ina heme group. The differences between those cytochromes liesin the different side-chains. For instance Cytochrome a has a heme a prosthetic group and cytochrome b has a heme b prostheticgroup. These differences result in different Fe2+/Fe3+ redoxpotentialssuch that various cytochromes are involved in themitochondrial electron transport chain.Cytochrome P450 enzymes perform the function of inserting anoxygen atom into a C—H bond, an oxidation reaction
RubredoxinRubredoxin is an electron-carrier found insulfur-metabolizing bacteria and archaea.The active site contains an iron ion whichis coordinated by the sulphur atoms offourcysteine residues forming an almostregular tetrahedron. Rubredoxins performone-electron transfer processes. Theoxidation state of the iron atom changesbetween the +2 and +3 states. In bothoxidation states the metal is high spin,which helps to minimize structuralchanges. rubredoxin active site
PlastocyaninPlastocyan is one of the family of bluecopper proteins which are involved in electrontransfer reactions. The copper binding site isdescribed as a ‘distorted trigonal pyramidal’.Thetrigonal plane of the pyramidal base is composedof two nitrogen atoms (N1 and N2) from separatehistidines and a sulfur (S1) from a cysteine. Sulfur(S2) from an axial methionine forms the apex.The ‘distortion’ occurs in the bond lengthsbetween the copper and sulfur ligands. Plastocyanin copper binding
Metal-ion storage and transferIronIron is stored as iron(III) in ferritin. The exact nature of thebinding site has not yet been determined. The iron appears tobe present as an hydrolysis product such as FeO(OH). Iron istransported by transferrin whose binding site consists of twotyrosines, one aspartic acid and one histidine The human bodyhas no mechanism for iron excretion. This can lead to iron-overload problems in patients treated with blood transfusions,as, for instance, with β-thallasemia.CopperCeruloplasmin is the major copper-carrying protein in theblood. Ceruloplasmin exhibits oxidase activity, which isassociated with possible oxidation of Fe2+ (ferrous iron) intoFe3+ (ferric iron), therefore assisting in its transport in theplasma in association with transferrin, which can only carryiron in the ferric state.
Metalloenzymes all have one feature in common, namely, that the metal ion is boundto the protein with one labile coordination site. As with all enzymes, the shape of theactive site is crucial. The metal ion is usually located in a pocket whose shape fits thesubstrate.Carbonic anhydrase Active site of carbonic anhydrase. The three coordinating histidine residues areshown in green, hydroxide in red and white, and the zinc in gray. CO2 + H2O H2CO3This reaction is very slow in the absence of a catalyst, but quite fast in the presence ofthe hydroxide ion CO2 + OH- HCO3-
Vitamin B12-dependent enzymesVitamin B12 catalyzes the transfer of methyl (-CH3) groupsbetween two molecules, which involves the breaking of C-Cbonds, a process that is energetically expensive in organicreactions. The metal ion lowers the activation energy for theprocess by forming a transient Co-CH3 bond.This is a naturallyoccurring organometallic compound, which explains itsfunction in trans-methylation reactions, such as the reactioncarried out by methionine synthase.
Nitrogenase (nitrogen fixation)The fixation of atmospheric nitrogen is a very energy-intensive process, as it involves breaking the very stable triple bond between the nitrogen atoms. The enzyme nitrogenase is one of the few enzymes that can catalyze the process. The enzyme occurs in certain bacteria.There are three components to its action:1. a molybdenum atom at the active site,2. Iron-sulfur clusters which are involved in transporting the electrons needed to reduce the nitrogen and3. an abundant energy source.The energy is provided by a symbiotic relationship between the bacteria and a host plant, often a legume. The relationship is symbiotic because the plant supplies the energy by photosynthesis and benefits by obtaining the fixed nitrogen.
Superoxide dismutaseThe superoxide ion, O2- is generated in biological systems by reduction ofmolecular oxygen. It has an unpaired electron, so it behaves as a free radical. It isa powerful oxidising agent. These properties render the superoxide ion very toxicand are deployed to advantage by phagocytes to kill invading micro organisms.Otherwise, the superoxide ion must be destroyed before it does unwanted damagein a cell. The superoxide dismutase enzymes perform this function veryefficiently. Oxidation: M(n+1)+ + O2− → Mn+ + O2 Reduction: Mn+ + O2− + 2H+ → M(n+1)+ + H2O2.This type of reaction is call a dismutation reaction. It involves both oxidation andreduction of superoxide ions. The superoxide dismutase group of enzymes,abbreviated as SOD, increase the rate of reaction to near the diffusion limitedrate.
CalmodulinCalmodulin is an example of a signal-transduction protein. It is a small proteinwhich contains four EF-hand motifs, each of which can bind a Ca2+ ion.In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidalconfiguration.The protein has two approximately symmetrical domains, separated by a flexible"hinge" region. Binding of calcium causes a conformational change to occur inthe protein. Calmodulin participates in an intracellular signalling system byacting as a diffusible second messenger to the initial stimuli.
The EF hand is a helix-loop-helix structural domain found in a large family ofcalcium-binding proteins. The EF-hand motif contains a helix-loop-helix topology,much like the spread thumb and forefinger of the human hand, in which theCa2+ ions are coordinated by ligands within the loop. It consists of twoalpha helices positioned roughly perpendicular to one another and linked by a short loop region(usually about 12 amino acids) that usually binds calciumions. The motif takes itsname from traditional nomenclature used in describing the protein parvalbumin,which contains three such motifs and is probably involved in muscle relaxation viaits calcium-binding activity. EF hands also appear in each structural domain of thesignaling protein calmodulin and in the muscle protein troponin-C.
Regulation and Control Metalloenzyme Inhibition•Approximately one-third of the known enzymes have metals as partof their structure, require that metals be added for activity, or arefurther activated by metals.•In enzymes where a metal has been built into the structure of theenzyme molecule, the metal cannot be removed without destroyingthat structure. Such enzymes include the metalloflavoproteins, thecytochromes, and the ferredoxins.•Metals resemble protons (H+) in that they are electrophiles that arecapable of accepting an electron pair to form a chemical bond. Indoing so, metals may act as general acids to react with anionic andneutral ligands. This characteristic of metals is helpful in enzymaticstructure and function but makes the enzyme it is part of pHdependent. Changes in pH can disrupt this electron flow that the metalwould normally help facilitate and thus inhibit the overalleffectiveness of the metalloenzyme.
ANTIOXIDANTAn antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules.Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells.
HISTORY• The term antioxidant originally was used to refer specifically to a chemical that prevented the consumption of oxygen. In the late 19th and early 20th century, extensive study was devoted to the uses of antioxidants in important industrial processes, such as the prevention of metal corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling of internal combustion engines.• Early research on the role of antioxidants in biology focused on their use in preventing the oxidation of unsaturated fats, which is the cause of rancidity. Antioxidant activity could be measured simply by placing the fat in a closed container with oxygen and measuring the rate of oxygen consumption. However, it was the identification of vitamins A, C, and E as antioxidants that revolutionized the field and led to the realization of the importance of antioxidants in the biochemistry of living organisms.
• The possible mechanisms of action of antioxidants were first explored when it was recognized that a substance with anti- oxidative activity is likely to be one that is itself readily oxidized.Research into how vitamin E prevents the process of lipid peroxidation led to the identification of antioxidants as reducing agents that prevent oxidative reactions, often by scavenging reactive oxygen species before they can damage cells.
GENERATION OF EXCESS FREE RADICALS • Faulty dietary habits • Diet high in animal fats • Sunlight • Toxic Drugs • Cigarette smoking • Over-exercise • Environmental Pollution
CLASSIFICATION OF ANTIOXIDANTS 1.) VITAMINS a) Vitamin E - It is fat soluble, alpha tocopherol is in a unique position to safeguard cell membranes largely composed of fatty acids from damage by free radicals. Alpha tocopherol also protects the fats in low density lipoproteins from oxidation.b)Vitamin C - It scavenge free radicals that are in an aqueous (watery) environment, such as inside cells. Vitamin C works synergistically with vitamin E to quench free radicals.c) Vitamin A -Vitamin A (retinol), also synthesized by the body from beta-carotene, protects dark green, yellow and orange vegetables and fruits from solar radiation damage, and is thought to play a similar role in the human body.
2.MINERALS : a) Selenium b) Manganese c) Copper d) ZincThese are components of antioxidant enzyme like glutathione peroxidase, superoxide dismutase and catalase.3.VITAMIN COFACTORS : a) Coenzyme Q104.CAROTENOIDS: a) Beta- carotene : It is the best quencher of single oxygen(an energized but uncharged form of oxygen that is toxic to cells). Beta- carotene is also especially excellent at scavenging free radicals in low oxygen peroxidase.b) Lycopenec) Lutein
5. a) Flavonoid polyphenolic • Flavones 7.Other nonflavonoid phenolics • Apigenin •Flavonolignans • Luteolin •Xanthones • Tangeritinb) Flavonols 8.Other Organic Antioxidants• Myricetin •Bilirubin• Proanthocyanidins •Citric acid •Lignanc) Flavanones: •R alphalipoic acid• Hesperetin •Uric acidd) Flavanols and their polymers The antioxidant enzymes are• Isoflavone phytoestrogens •Superoxide dismutase,• Anthocyanins •Catalase •Glutathione peroxidase6. Phenolic acids and their esters They serve as primary line of • Chlorogenic acid defense in destroying free radicals. • Ellagic acid • Gallic acid • Rosmarinic acid
Other Classifications1.Preventive Antioxidants :- They inhibit the initial production of free radicals. They include catalase,glutathione peroxidase,diethyltriamine pentaacetate and ethylene diamine tetra-acetate(EDTA).2.Chain breaking Antioxidants :- They inhibit the damaging phase of free radicals. They include superoxide dismutase, uric acid and Vitamin E. Alpha tocopherol act as the most effective naturally occurring chain breaking antioxidants in body tissues.3.Water soluble antioxidants:- Water soluble antioxidants are referred to as hydrophilic antioxidants. Basically, they are able to assist the body in the process of cell cytosol and help out in the blood plasma. In other words they take a hands on approach to ridding the body of harmful free radicals and pollutants. Most common water soluble antioxidants are:• Ascorbic acid• Glutathione• Lipoic acid• Uric acid
4 .Lipid Soluble Antioxidants:Unlike water soluble antioxidants, the lipid soluble version do not actively go out seeking to destroy rogue cells in the human body. These are the antioxidants which in fact have a much more passive role in keeping the human body healthy. Basically, these antioxidants work by clinging on to damaged cells, and injecting valuable nutrients which support the replenishment and health of that particular individual cell. In this way, antioxidants are able to promote the health of cells on a celllular level,by working in sync with the cells themselves.For e.g. :- Carotenes , Ubiquinol Deficiency of antioxidantsA shortage of antioxidants could cause, or assist in causing Alzheimers disease, cancer, cardiovascular disease, cataracts, diabetes, hypertension, infertility, macular degeneration (eye lens degeneration) , mental illness, respiratory tract infection.By adding enough antioxidants to the diet, there is less oxidation stress, and aging is also slowed down.
FOOD SOURCES OF ANTIOXIDANTS• Beta-carotene- Green vegetables, ripe yellow fruits & vegetables like papaya ,mango ,pumpkin and carrots.• Vitamin A – Milk fat, Egg yolk,liver,kidney & fortified vanaspati.• Vitamin C – Fresh & Citrus fruits- amla,orange,lemon,sweet lime,guava and gooseberry, green leafy vegetables & sprouted pulses.• Vitamin E – Oil seeds,cereals,nuts,cereal products, vegetable oils & egg yolk.• Selenium & Zinc – Meat,Sea-food,Cereals & pulses.• Copper – Oysters, Mushroom, Liver & nuts.• Iron- Green leafy vegetables, cereals, millets,pulses,nuts,meat and liver
Non-Nutrient antioxidants sources:Flavonoid, flavonols, phenolic acids, non flavonoid phenolics. Such rich sources of these compounds are bean, cloves, oats, tea, coffee, grapes, turmeric, mustard,walnut, tomato, brown rice, oak bark, red wine.
Antioxidants and agingAging is an irreversible phenomenon for all living organisms. With aging, cell division and replacement of dead or damaged cells slows down. Cell death, mutation or damage is partly caused by the free radicals.Free radicals affect the skin in three main ways:• They can alter the fatty layers in cellular membranes. These fatty layers provide structure to the cell, and control which nutrients and other agents can pass in and out.• They can alter the DNA within cells, which aside from the potential to develop into serious illnesses, can make skin inclined to wrinkles and sagging before its natural biological time.• Altered DNA creates a blueprint for collagen and elastin fibers that dont function as healthy, normal ones would.
Free radicals also lead to a process called the cross-linking of collagen fibers. This occurs in the skins dermis, as a result of collagen and elastin fibers becoming hard, thick, and then binding together. Cross-linked fibers create wrinkles, skin sag, and cause regular expression lines to become etched in face as a permanent fixture.With healthy collagen and elastin fibres these expression lines would simply disappear once moved facial muscles in a different way. And enzymes that metabolize collagen are encouraged by free radicals, which, given the importance of collagen in youthful looking skin, is best minimized. Antioxidant and aging relationship• They strengthen the capillaries that supply important nutrients to the skin cells, as well as supporting cellular membranes.• Healthy cell membranes regenerate quickly and slow the aging process.• Antioxidants anti-aging benefit is due to their anti-inflammatory effect.
ANTIOXIDANT AND CANCERCancer – a disease that affects so many around the world and continues to be studied earnestly in order to finally identify a cure. But, in the meantime, researchers, in an effort to take control of the spread of this disease, promote programs of prevention. Diet, exercise, and the avoidance of controllable environmental pollutants are all part of the effort to prevent cancer.It has been shown that cancer derives from good cells gone bad. Affected by poor diet, environmental factors, and chemical substances, molecules inside the body lose electrons in response. The molecules become free radicals and, as such, they begin their attack on healthy cells to take back electrons.
Antioxidants and cancer:• Antioxidants bolster the immune system and work alongside healthy cells to combat free radicals.• Cancer works against the cells in the body while antioxidants work on behalf of cells.Studies related to antioxidants and cancer:• The first large randomized trial on antioxidants and cancer risk was the Chinese Cancer Prevention Study, published in 1993. This trial investigated the effect of a combination of beta-carotene, vitamin E and selenium significantly reduced incidence of both gastric cancer and cancer overall.• A 1994 cancer prevention study entitled the Alpha- Tocopherol/Beta Carotene Cancer Prevention Study demonstrated that lung cancer rates of Finish male smokers increased significantly with beta-carotene and were not affected by vitamin E.
ANTIOXIDANT AND DIABETESDiabetes is a metabolic disorder. The disorder is due to a deficiency or diminished effectiveness of the hormone insulin.Antioxidants are beneficial for diabetes suffers, not only to maintain antioxidant levels in the body but also to treat the long term complications that can arise.• Neuropathy• Retinopathy• NephropathyMultiple sources of oxidative stress in diabetes including -• Non enzymatic,• Enzymatic• Mitochondrial pathways.
Antioxidants and Cardiovascular DiseasesAtherosclerosis is a condition where the walls of the arteries are damaged and narrowed by deposits of cholesterol and other fatty substances, calcium, fibrin, and cellular wastes , eventually blocking off the flow of blood. High blood levels of cholesterol - particularly the cholesterol carried by low- density lipoprotein ("LDL", a protein found in blood) - are associated with an increased risk of atherosclerosis.Oxidation of LDL is believed to contribute to the development of atherosclerosis (Frei 1995). Macrophage cells preferentially take up oxidized LDL, become loaded with lipids, and convert into "foam cells" (Aviram 1996). Foam cells accumulate in fatty streaks, early signs of atherosclerosis. Humans produce auto-antibodies against oxidized LDL.
The identification of LDL oxidation as a key event in atherosclerosis suggests that it may be possible to reduce the risk of atherosclerosis by antioxidant supplementation (Ylä- Herttuala 1991). Vitamin E is the major naturally-occurring antioxidant in human lipoproteins (Bowry et al. 1992). Most circulating carotenoids are associated with lipoproteins in plasma (Clevidence and Bieri 1993). Bieri 1993).The largest fraction of total carotenoids is found in LDL, as evidenced by the typically yellow color of this lipoprotein fraction (Clevidence and Bieri 1993). The largest fraction of hydrocarbon carotenoids (e.g., beta-carotene and lycopene), as well as most vitamin E and other tocopherols, is transported by LDL ( Oshima et al. 1997), suggesting that these compounds in particular may play an important role in preventing oxidative modification of this lipoprotein fraction.
REFERENCE•Chatterjea M.N., Shinde,Rana, Textbook of MedicalBiochemistry. Jaypee Brothers Medical Publishers, NewDelhi,1999.•Deb,A.C., Fundamentals of Biochemistry. New Central Bookagency(P)Ltd., Kolkata, 2008.•http://en.wikipedia.org/wiki/Metalloprotein•http://depts.washington.edu/chemcrs/bulkdisk/.../notes_Lecture_3.pdf•www.sciencemag.org/content/261/5122/701.full.pdf•http://en.wikipedia.org/wiki/Antioxidant