Vitamins PDF - Enzymes and Cofactors

Vitamins and Cofactors* Assoc. Prof. Victor Markus, PhD Department of Medical Biochemistry Near East University, Faculty of Medicine *Prepared by Prof. Dr. Özlem Dalmızrak, Dept of Medical Biochemistry, Near East University.  Outline Introduction: Enzymes and cofactors. Stru...

Vitamins and Cofactors* Assoc. Prof. Victor Markus, PhD Department of Medical Biochemistry Near East University, Faculty of Medicine *Prepared by Prof. Dr. Özlem Dalmızrak, Dept of Medical Biochemistry, Near East University.  Outline Introduction: Enzymes and cofactors. Structure and function of Vitamins Coenzymes and Cofactors  ENZYMES The organism must be able to self-replicate It must be able to catalyze chemical reactions efficiently and selectively. Enzymes are central to every biochemical process. – They have a high degree of specificity for their substrates. – They accelerate chemical reactions tremendously. – They function in aqueous solutions under very mild conditions of temperature and pH. – All enzymes are proteins. Except small group of catalytic RNA molecules called ribozymes (Example, Ribosome and Ribonuclease P (RNase P)). – Their catalytic activity depends on the integrity of their native protein conformation. – Molecular weights ranging from 12 000 to more than 1 million Dalton. – Some enzymes do not need any chemical groups for activity other than their amino acid residues. Others require an additional compound called a cofactor.  Cofactors are either one or more inorganic ions, such as Fe2+, Mg2+, Mn2+, Zn2+, or a complex organic or metalloorganic molecule called coenzyme. Coenzymes act as transient carriers of specific functional groups. A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme is called a prosthetic group. A catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme. The protein part of such an enzyme is called the apoenzyme or apoprotein.  Enzymes are classified according to the reactions they catalyze. Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word describing their activity. Urease → hydrolysis of urea DNA polymerase → polymerization of nucleotides to form DNA Pepsin → Greek “pepsis” = “digestion” Lysozyme → lyse bacterial walls  The same enzyme may have two or more names or two different enzymes may have the same name. – Alanine aminotransferase (transaminase) (ALT) = Serum glutamic pyruvic transaminase (SGPT) Enzyme Commission (EC) Number: ATP + D-Glucose → ADP + D-Glucose 6-phosphate Enzyme: ATP:glucose phosphotransferase (E.C. 2.7.1.1.) (2) Class: transferase (7) Subclass: phosphotransferase (1) Phosphotransferase with a hydroxyl group as acceptor (1) D-glucose as the phosphoryl group acceptor. More commonly used name : Hexokinase  International Classification of Enzymes Class No Class Name Type of reaction catalyzed 1 Oxidoreductases Transfer of electrons (hydride ions or H atoms) 2 Transferases Group transfer reactions 3 Hydrolases Hydrolysis reactions (transfer of functional groups to water) 4 Lyases Addition of groups to double bonds, or formation of double bounds by removal of groups 5 Isomerases Transfer of groups within molecules to yield isomeric forms 6 Ligases Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor  Classification 1. Oxidoreductases 2. Transferases 3. Hydrolases The graph displays the proportion of 4. Lyases genes belonging to each enzyme class 5. Isomerases 6. Ligases  How enzymes work? Active site is a place on the enzyme where the enzyme- catalyzed reaction takes place. The molecule that is bound in the active site and acted upon by the enzyme is called the substrate. E+S ES EP E+P Enzymes decrease the energy of activation for a reaction. (They do not affect the equilibrium concentrations of the substrates and products, nor do they change the overall Gibbs free energy change (ΔG) for the reaction.) Cofactors are required to convert inactive apoenzymes to active holoenzymes Apoenzyme + Cofactor = Holoenzyme (Inactive) (Active)  Essential Ions More than 25% of all known enzymes require metallic cations to achieve full catalytic activity. 1. Metal activated enzymes either have an absolute requirement for added metal ions or are stimulated by the addition of metal ions. Monovalent cation: K+ Divalent cations: Ca2+, Mg2+ 2. Metalloenzymes contain firmly bound metal ions at their active sites. The most common metals are iron and zinc, and less often copper and cobalt. Carbonic anhydrase (erythrocytes) CO2 + H2O  H+ + HCO3- Some Inorganic Elements That Serve as Cofactors for Enzymes  Coenzymes Coenzymes can be classified into two types according to how they interact with the apoenzyme. 1. Cosubstrates are substrates in enzyme-catalyzed reactions. 2. Prosthetic group remains bound to the enzyme during the reaction. Cosubstrates and prosthetic groups are part of the active site of the enzyme. Prokaryotes, protists, fungi and plants are capable of synthesizing their coenzymes from simple precursors. But animals (including human) can not!!!! We need a source of coenzymes or their intermediate precursors in order to survive.  STRUCTURE AND FUNCTION OF VITAMINS Mammals and other animals rely on other organisms to supply these micronutrients. These essential compounds are called vitamins. The ultimate sources of vitamins are usually plants and microorganisms, though carnivorous animals can obtain vitamins from meat. Some vitamins are found in foods as provitamins and they are activated in body. Some of them can be synthesized endogenously (Vitamin D from 7-dehydrocholesterol; niacin from tryptophan and vitamin K synthesis in intestinal flora)  Suggested daily amounts of vitamins for adults are called recommended dietary allowance (RDA) or dietary reference intake (DRI). This recommended amount might be different according to the gender and age. RDA (or DRI) is determined by international health organizations and defined as “the amount of vitamin that can protect the body against illness derived from any vitamin deficiency”. Units for vitamin concentration: mcg (µg) and IU (International Unit)  A nutritional-deficiency disease (Avitaminoses) can occur when a vitamin is deficient or absent in the diet of an animal. Causes: Some Vitamins and Their 1. Malnutrition Associated Nutritional-deficiency 2. Oral contraceptives Diseases 3. Smoking 4. Pregnancy 5. Lactation 6. Infection (B3) 7. Intestinal system disorders 8. Long term drug treatment (B5) Symptoms: 1. Decrease in tissue concentration (B7) 2. Change in biochemical parameters 3. Anatomic lesion (B9) 4. Clinical symptoms start Vit D3 Rickets  Classification Water-soluble vitamins (such as B vitamins) Fat-soluble vitamins (lipid vitamins) (A, D, E, K) Common features of water soluble vitamins Excreted with urine, no sign of hypervitaminose Except B12, they are not stored Except B12, they are found in herbal products Sensitive to heat Most of them act as a coenzyme  Water-soluble vitamins (such as B vitamins) Fat-soluble vitamins (lipid vitamins) (A, D, E, K) Common features of fat soluble vitamins All apolar and hydrophobic All contain isoprene units Fully absorbed when absorbtion of fats is enough In blood, they are carried with lipoproteins and specific carrier proteins They can be stored in body. Overdose can cause toxicity Major storage organ is liver (Long term storage) Defects in digestion, absorbtion and deficiency in pancreatic secretion  Symptoms arise They do not act as a coenzyme Resistant to heat Generally found in animal products (meat, milk, egg) They have different functions  Water-soluble vitamins Vitamin B1 (Thiamine) Cofactor: Thiamine pyrophosphate (TPP) Thiamine is synthesized in bacteria, fungi, and plants. Animals must obtain it from their diet. Insufficient intake in mammals results in a disease called beriberi affecting the peripheral nervous system and/or the cardiovascular system, with fatal outcome if not cured by thiamine administration. Dietary source: Yeast, unpolished rice, meat, potatoes, whole grains, nuts Recommended Daily Allowance : 1.4 mg Function: Thiamine is a coenzyme in energy producing reactions especially in carbohydrate metabolism. As a TPP it involes in oxidative decarboxylation of α-keto acids: – Pyruvate dehydrogenase – α-ketoglutarate dehydrogenase – Transketolase in pentose phosphate pathway (PPP) TPP located in the peripheral nerve membranes are responsible for the acetylcholine synthesis and phosphorylation of sodium transport channel. Deficiency: Pyruvate accumulates and pentose phosphate pathway (PPP) stops. Absence of energy and NADPH affects cells directly. Due to role of TPP in neurotransmission, symptoms in nervous system occurs. Early symptoms: Loss of apetite, constipation, nausea, depression, peripheric neuropathy and irritability. Advanced symptoms: mental convulsion, ataxia, opthalmoplegia These symptoms are accompanied with the Wernicke-Korsakoff syndrome observed in chronic alcoholics. This situation is multifactorial. Insufficient vitamin intake and intestinal absorbtion, inhibitory effect of alcohol and insufficient metabolism in liver can lead Wernicke encephalopathy and Korsakoff psycosis. Vitamin B2 (Riboflavin) Cofactor: FMN, FAD Dietary source: Liver, rice, whole grain cereals, egg, milk Recommended Daily Allowance : 1.2 mg Deficiency: In hypothyroidism, conversion of riboflavin to FAD and FMN is slow. Requirement increases during development, pregnancy and in hyperthyroidism. Glossitis (inflamation of the tongue), stomatitis (inflamation of the mouth) and dermatitis Vitamin B3 (Niacin) Cofactor: NAD, NADP Dietary source: Meat, liver, fish, milk, eggs, broccoli, carrots Recommended Daily Allowance : 18 mg Resistant to heat and light. Small amounts of niacin can be stored in liver. Some disease, pregnancy, stress and exercise increase the requirement. Deficiency: Pellagra which is characterized by diarrhea, dermatitis, and dementia as well as “necklace” lesions on the lower neck, hyperpigmentation, thickening of the skin, inflammation of the mouth and tongue, digestive disturbances, amnesia, delirium, and eventually death, if left untreated. Vitamin B5 (Pantothenic acid) It contains β-alanine and pantoic acid. Cofactor: Coenzyme A Acyl carrier protein (ACP) Deficiency: Rare Dietary source: Liver, kidney, nuts, egg yolk, fish, chicken Recommended Daily Allowance : 5 mg Vitamin B6 (Pyridoxine) Cofactor: Pyridoxal phosphate Amino acid metabolism (transamination, decarboxylation), synthesis of sphingolipids. Coenzyme of phosphorylase (glycogen break-down) Deficiency: dermatologic and neurologic changes Dietary source: meats, whole grain products, vegetables, nuts, liver, kidney, fish Recommended Daily Allowance : 1.3 mg Vitamin B7 (Biotin) (Vitamin H) Cofactor: Biotin Coenzyme of carboxylase enzymes. CO2 carrier. Deficiency: Neurological symptoms in adults such as depression Dietary source: Common in natural products and also synthesized by intestinal bacteria. Beans, breads, brewer's yeast, cauliflower, chocolate, egg yolks, fish, kidney, legumes, liver, meat, molasses, dairy products, nuts, oatmeal, oysters, peanut butter, poultry, wheat germ, and whole grains. Recommended Daily Allowance : 30-100 µg Vitamin B9 (Folic acid) Cofactor: Tetrahydrofolate Tetrahydrofolate carries one-carbon compounds like methyl, methylene, formyl. Deficiency: neural tube defects in developing embryos and megaloblastic anemia Dietary source: Leafy green vegetables, whole grain cereals, liver, kidney Recommended Daily Allowance : 400 µg Vitamin B12 Cofactor: Cobalamin Dietary source: Meat, fish, eggs, dairy products, yeast, synthesized by intestinal bacteria. Deficiency: As a result of decrease in secretion of intrinsic factor (IF) and/or HCl, long term vegeterian diet and malabsorbtion, B12 deficiency occur. Pernicious anemia, megaloblastic anemia Recommended Daily Allowance : < 1000 µg In stomach, cobalamin is separated from digested foods by pepsin and HCl and attached salivary protein, haptocorrin (HC). In duedonum, vitamin is released by pancreatic enzymes. Intrinsic factor (IF) is a glycoprotein secreted by parietal cells of gastric mucosa. In ileum IF binds B12. This complex is taken into the ileum epihelial cells via cubilin (CB) receptors. In cells IF- cobalamin complex dissociates and transcobalamin II (TCII) binds B12. B12-TCII complex is called holotranscobalamin. Holotranscobalamin is released to plasma. This complex is taken into the cells (liver and other tissues) via TCII receptors (TCIIR). Vitamin is released from TCII and found in cytosol as methylcobalamin in mitochondria as 5’- desoxyadenosylcobalamin.  Vitamin C: Vitamin but not a coenzyme Ascorbic acid acts as a reducing agent during the hydroxylation of collagen. Dietary Source: Tomatoes, potatoes, fruits and green vegetables. Deficiency: Scurvy disease (as a result of abnormal collagen synthesis)  Lipid Vitamins (Fat-Soluble Vitamins) Structure of the four lipid vitamins (A, D, E, and K) contain rings and long aliphatic side chains. Highly hydrophobic, although each possesses at least one polar group. Ingested lipid vitamins are absorbed in the intestine. After digestion of any proteins that may bind them, they are carried to the cellular interface of the intestine as micelles formed with bile salts. Vitamin A (Retinol): 20-carbon lipid molecule obtained in the diet either directly or indirectly from β-carotene. Retinoic acid: Signal compound that binds to receptor proteins inside cells; the ligand-receptor complexes then bind to chromosomes and regulate gene expression during cell differentiation. Retinal: Light-sensitive compound with an important role in vision. Retinal is a prosthetic group of the protein rhodopsin. Absorbsion of a photon of light by retinal triggers a neural impulse. Dietary source for vitamin A: Oily fish, liver, eggs, whole milk and butter Dietary source for β-carotene: Carrots, sweet potatoes, and other yellow vegetables. RDA: 5000 IU (3 mg β-carotene) (man), 4000 IU (2-4 mg β-carotene) (woman). Toxicity over 15000 IU. Deficiency: Dryness of the skin, eyes and mucous membranes Retarded development and growth Night blindness Tendency to inflammation Toxicity: When amount of vitamin A exceeds the capacity of retinol- binding protein, it accumulates in tissues and causes tissue damage. Toxicity primarily affects central nervous system and results in headache, sickness, ataxia, anorexia. Hepatomegalia, histological changes in liver, hyperlipidemia, thickness in long bones, hypercalcemia, dryness in skin are the other symptoms of Vitamin A deficiency. Excess usage in pregnancy may affect the fetus development and cause malformations. Vitamin D: Sterol derivative. It is taken as a provitamin. It has two forms: Plant-derived ergocalciferol and animal-derived cholecalciferol. Regulates both intestinal absorbtion of calcium and its deposition in bones. Main source is action of sunlight on the skin. Deficiency: Defective bone formation Rickets (in children) Osteomalacia (in adults) Dietary Source: Dairy products, oily fish, egg yolk RDA: 5 µg cholecalciferol or 200 IU Vitamin D Toxicity: Increased Ca absorbtion and bone resorption can cause hypercalcemia and metastatic calsification. Hypercalcemia, calciuria and kidney stones. Vitamin E (α-tocopherol): Function as a reducing agent that removes oxygen and free radicals. This antioxidant action may prevent damage to fatty acids in biological membranes. RDA: 10 mg (man), 8 mg (woman) Dietary Source: Eggs Liver Vegetable oil Wheat  Concentration of Vitamin E is especially high in erythrocytes and retina. Absorbtion: From intestines Vitamin E is first transferred to lymphoid system then liver. Most of the Vitamin E is found in fat tissue, liver, heart, muscle, testicles, uterus, adrenal gland and brain. In plasma it is carried with lipoproteins. Deficiency: A deficiency of vitamin E is rare but may lead to fragile red blood cells and neurological damage. Toxicity: Vitamin E is the least toxic among all fat soluble vitamins. Excess amounts can be excreted by urine and feces. High doses can cause nausea and diarrhea. Vitamin K (Phylloquinone): It is a lipid vitamin from plants that is required for the synthesis of some of the proteins involved in blood coagulation. Source: Bacteria in large intestine. Dietary source: Green vegetables, soya beans, potatoes, meat, green tea. Vitamin K deficiency is not common. Toxicity: Vitamin K has a toxic effect on erythrocyte membranes. In infants long term vitamin K treatment can cause hemolytic anemia and jaundice.  When calcium binds to the -carboxyglutamate residues of the coagulation proteins, the proteins move to the platelets surfaces where many steps of the coagulation process take place. Warfarin is a synthetic compound that inhibits the formation of active prothrombin. It is an anticoagulant drug for treating humans at risk for excessive blood clothing.  COENZYMES 1. ATP and Other Nucleotide Cosubstrates Nucleoside triphosphates: Adenosine triphosphate (ATP) Guanosine triphosphate (GTP) S-adenosinemethionine (SAM) Nucleotide sugars such as uridine diphosphate glucose (UDP- glucose  ATP ATP can donate its phosphoryl, pyrophosphoryl, adenylyl (AMP) or adenosyl groups in group transfer reactions. ATP is also the source of several other metabolite coenzymes.  S-adenosinemethionine (SAM) Methionine + ATP → S-adenosinemethionine + Pi + PPi SAM reacts with nucleophilic acceptors and is the donor of almost all the methyl groups used in biosynthetic reactions. Methylation reactions that require SAM include methylation of phospholipids, proteins, DNA and RNA. In plants, SAM is involved in regulating the ripening of fruit (Precursor of the plant hormone ethylene). Nucleotide-sugar coenzymes Glycogen Synthesis  2. Nicotinamide coenzymes Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+) They play a role in many oxidation-reduction reactions. They assist in the transfer of electrons to and from metabolites. NAD+ and NADP+ almost always act as a cosubstrates for dehydrogenases. Pyridine nucleotide-dependent dehydrogenases catalyze the oxidation of their substrates by transferring two electrons and a proton in the form of a hyride ion (H-) to C-4. Oxidation of pyridine nucleotides occurs two electrons at a time. NAD(P)+ + 2e- + 2H+ NAD(P)H + H+ Most reactions forming NADH and NADPH are catabolic reactions. The oxidation of NADH in mitochondia is coupled to the synthesis of ATP (Electron transport chain). Most NADPH is used as a reducing agent in biosynthetic reactions. In most tissues the total concentration of; NAD+ + NADH 10-5 M NAD+ / NADH is high. NADPH >> NADP+ NAD+ generally functions in oxidations-usually as a part of a catabolic reaction. NADPH is the usual coenzyme in reductions-as a part of an anabolic reaction.  3. FAD and FMN Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derived from riboflavin (vitamin B2). Riboflavin FMN: black; FAD: black and blue Flavoenzymes: Oxidoreductases require FMN or FAD as a prosthetic group. The prosthetic group is very tightly bound, usually noncovalently. FAD and FMN are reduced to FADH2 and FMNH2 by taking up a proton and two electrons in the form of hydride ion. FADH2 and FMNH2 donate electrons either one or two at a time. A partially oxidized compound FAD· or FMN· is formed when one electron is donated.  4. Coenzyme A (CoA or HS-CoA) CoA has a role in; Oxidation of fuel molecules Biosynthesis of some carbohydrates and lipids CoA is involved in acyl-group transfer reactions.  5. Thiamine Pyrophosphate (TPP) TPP is synthesized from thiamine (vitamin B1) by enzymatic transfer of pyrophosphoryl group from ATP. About half a dozen decarboxylases require TPP as a coenzyme. TPP is also a coenzyme involved in the oxidative decarboxylation of -keto acids.  6. Pyridoxal Phosphate (PLP) The B6 family of water-soluble vitamins consist of three closely related molecules that differ only in the state of oxidation and amination of the carbon bound to position 4 of the pyridine ring. PLP is a prosthetic group for many enzymes that catalyze a variety of reactions involving amino acids, including isomerizations, decarboxylation, and side-chain eliminations or replacements. Removal of the -amino group from amino acids is catalyzed by transaminases which participate in both the biosynthesis and degredation of amino acids.  7. Biotin Biotin is a prosthetic group for enzymes that catalyze carboxyl- group transfer reactions and ATP-dependent carboxylation reactions. Because biotin is synthesized by intestinal bacteria and is required only in very small amounts, biotin deficiency is rare in humans or animals fed with normal diets.  Reaction catalyzed by pyruvate carboxylase: Pyruvate + HCO3- + ATP → Oxaloacetate + ADP + Pi  8. Tetrahydrofolate Folate has three main components: 1.Pterin (2-amino-4-oxopteridine) 2.p-amino benzoic acid moiety 3.Glutamate residue Humans require folate in their diet bacause we cannot synthesize the pterin-p-aminobenzoic acid intermediate (PABA) and we cannot add glutamate to exogenous PABA The coenzyme form of folate known as tetrahydrofolate. n = 5-6 Enzyme: Dihydrofolate reductase The primary metabolic function of dihydrofolate reductase is the reduction of dihydrofolate produced during the formation of the methyl group of dTMP. This reaction, which uses a derivative of tetrahydrofolate, is an essential step in the biosynthesis of DNA. Dihydrofolate reductase has been extensively studied as a target for chemotheraphy in the treatment of cancer. One carbon derivatives of tetrahydrofolate 5,6,7,8-Tetrahydrobiopterin is a pterin coenzyme, but is not derived from a vitamin. It is synthesized by animals and other organisms. It is the cofactor for several hydroxylases and is a reducing agent in the conversion of phenylalanine to tyrosine.  9. Cobalamin (Vitamin B12) Corrin ring system Cobalamin is synthesized by some species of bacteria. It is required as a micronutrient by all animals and by some bacteria and algae. Humans obtain vitamin B12 from foods of animal origin. Deficiency of cobalamin can lead to pernicious anemia. It is a potentially fatal disease in which there is a decrease in the production of blood cells by bone marrow. Pernicious anemia can also cause neurological disorders. Most patients with pernicious anemia do not secrete a necessary glycoprotein (called intrinsic factor) from the stomach mucosa. This protein specifically binds cobalamin, and the cobalamin- intrinsic factor complex is absorbed by the cells of small intestine. Impaired absorbtion of cobalamin is now treated by regular injections of the vitamin. Methylcobalamin participates in the transfer of methyl groups. Adenosylcobalamin participates in several enzyme-catalyzed intramolecular rearrangements in which a hydrogen atom and a second group bound to adjacent carbon atoms within a substrate exchange places.  10. Lipoamide The cofactor lipoamide is the protein-bound form of lipoic acid. This structure is found in protein components of pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex.  Ubiquinone (Coenzyme Q) Ubiquinone transports electrons between membrane-embedded enzyme complexes in mitochondria inner membrane. Some bacteria use menaquinone. Plastoquinone (plants) serves a similar function in photosynthetic electron transport in chloroplasts. Ubiquinone is a stronger oxidizing agent than either NAD+ or the flavin coenzymes. It can be reduced by NADH or FADH2. Like FMN and FAD, ubiquinone accepts or donates two electrons one at a time because it has three oxidation states.  Protein Coenzymes (Group Transfer Proteins) They contain a functional group either as a part of their protein backbone or as a prosthetic group. Generally smaller and more heat-stable than most enzymes. Participate in group-transfer reactions or in oxidation-reduction reactions. Metal ions, iron-sulfur clusters and heme groups are commonly found in these protein coenzymes.  Cytochromes Cytochromes are heme-containing protein coenzymes whose Fe(III) atoms undergo reversible one-electron reduction. Cytochrome c (B5)