‹#› 1 Enzymes I Ó Department of Biochemistry, FM MU, 2013 (J.D.) General features, cofactors ‹#› 2 Literature for Biochemistry I •Lecture files on is.muni.cz. •Tomandl J., Táborská E.: Biochemistry I – Seminars. MU, 2012 •Harvey R.A., Ferrier D.R.: Biochemistry. 5th ed., Lippincott Williams & Wilkins, 2011. •Koolman J., Röhm K.H.: Color atlas of biochemistry, Thieme, 2013. • ‹#› 3 General features of enzymes •biocatalysts •different types of proteins / also RNA (ribozyme) • with covalently attached prosthetic group and/or metal cation, • oligomeric / multienzyme complexes / associated with membranes etc. •different distribution in cell and in the body, make isoforms (isoenzymes) •specific (towards substrate and reaction), highly effective •work under mild conditions •in vivo - can be regulated in two ways (activity of enzyme, quantity of enzyme) •in vitro - sensitive to many factors CAUTION: peptidyltransferase is ribozyme (AK)n-tRNA1 + AK-tRNA2 ® tRNA1 + (AK)n+1-tRNA2 rRNA ‹#› 4 Enzymes are highly efficient catalysts •decrease activation energy Þ increase the reaction rate •much more efficient than other (inorganic) catalysts •remain unchanged after reaction •do not alter equilibrium constant K •in vitro sensitive to many factors • ‹#› 5 Enzymes work under mild conditions •narrow temperature range around 37 °C •over 50 °C become denaturated = inactivated •narrow pH range Þ pH optimum •most intracellular enzymes have pH optima around 7 •digestion enzymes function in rather stronger acidic / alkaline environment (pepsin 1-2, trypsin ~ 8) ‹#› 6 Dual specifity of enzymes towards: •Reaction •catalyze just •one type of reaction • • •Substrate •work with one substrate •(or group of similar substrates) •often stereospecific ‹#› 7 Enzymes are stereospecific catalysts •there are two types of stereospecific conversions: 1.non-chiral substrate ® chiral product(one enantiomer) pyruvate ® L-lactate fumarate ® L-malate 2.chiral substrate(one enantiomer) ® product • L-alanine ® pyruvate (D-alanine does not react) • D-glucose ® ® pyruvate (L-glucose does not react) • chiral signal molecule ® complex with receptor ® biological response • chiral drug(ant)agonist ® complex with receptor ® pharmacological response 1. ‹#› 8 L-Lactate C COOH CH3 H HO H C C CH3 O O HO Enzyme Hydrogenation of pyruvate when pyruvate is hydrogenated without enzyme (in vitro), reaction product is the racemic mixture of D-lactate and L-lactate: in reaction catalyzed by lactate dehydrogenase (in vivo), pyruvate is reduced stereospecifically to L-lactate only: L-Lactate D-Lactate 1. non-chiral substrate ® chiral product ‹#› 9 Non-enzymatic hydration of fumarate in vitro reaction proceeds to racemic D,L-malate fumarate L-malate D-malate addition from one side addition from another side 1. non-chiral substrate ® chiral product ‹#› 10 Enzymatic hydration of fumarate (citrate cycle) in vivo just one enantiomer (L-malate) is produced reagent substrate 1. non-chiral substrate ® chiral product enzyme ‹#› 11 Hydrogenation of D-fructose in vitro gives two epimers 2 H + D-fructose D-glucitol D-mannitol reaction site is planar in vivo: enzymatic reaction gives just one product (D-glucitol) 1. non-chiral substrate ® chiral product ‹#› 12 Enzymes or receptors recognize only one enantiomer If the reactant of enzymatic reaction is a chiral compound, only one of two enantiomers is recognized as the specific substrate. R R X x proper enantiomer not-fitting enantiomer Chiral substrates/signal molecules are bound to the stereospecific enzymes/receptors at three sites: 2. chiral substrate ® product see also MCH II, p. 13 ‹#› 13 Enzyme nomenclature: the ending -ase Systematic names identify the enzymes fully with the EC code number, contain information about substrate and type of reaction, not very convenient for everyday use. Recommended (accepted) names are shorter than systematic names, include also some historical names (pepsin, amylase) EC (abbr. Enzyme Commission) of International Union of Biochemistry (IUB) major class number . subclass number . sub-subclass number . enzyme serial number http://www.chem.qmul.ac.uk/iubmb/enzyme/ ‹#› 14 Examples of enzyme names •Recommended name: alcohol dehydrogenase •Systematic name: EC 1.1.1.1 ethanol:NAD+-oxidoreductase •Reaction: ethanol + NAD+ ® acetaldehyde + NADH + H+ Recommended name: alanine aminotransferase (ALT) Systematic name: EC 2.6.1.2 L-alanine:2-oxoglutarate-aminotransferase Reaction: L-alanine + 2-oxoglutarate ® pyruvate + L-glutamate ‹#› 15 Classification of enzymes: six classes according to reaction type (each class comprises other subclasses) Enzyme class General scheme of reaction 1. Oxidoreductases Ared + Box D Aox + Bred 2. Transferases A-B + C ® A + C-B 3. Hydrolases A-B + H2O ® A-H + B-OH 4. Lyases A-B D A + B (reverse reaction: synthases) 5. Isomerases A-B-C D A-C-B 6. Ligases (synthetases) A + B + ATP ® A-B + ADP + Pi ‹#› 16 1 Oxidoreductases •catalyze the oxidation or reduction of substrate • •subclasses: •dehydrogenases catalyze the transfers of two H atoms •oxygenases catalyze the incorporation of one/two O atoms into the substrate (monooxygenases, dioxygenases) •oxidases catalyze transfers of electrons between substrates • (e.g. cytochrome c oxidase, ferroxidase) •peroxidases catalyze the decomposition of peroxides Example: lactate + NAD+ D pyruvate + NADH + H+ Recommended name: lactate dehydrogenase Systematic name: (S)-lactate:NAD+ oxidoreductase ‹#› 17 2 Transferases •catalyze the transfer of a group from one to another substrate •subclasses: •aminotransferases, methyltransferases, glucosyltransferases •kinases phosphorylate substrate by the transfer • of phosphoryl group PO32– from ATP (e.g. hexokinases, protein kinases) Example: glucose + ATP ® glucose 6-P + ADP Recommended name: glucokinase Systematic name: ATP:D-glucose phosphotransferase ‹#› 18 Example: Phosphorylation of glucose glucose glucose 6-phosphate glucokinase ‹#› 19 Example: glucose 6-P + H2O ® glucose + Pi Recommended name: glucose 6-phosphatase Systematic name: glucose 6-phosphate phosphohydrolase 3 Hydrolases catalyze the hydrolytic splitting of esters, glycosides, amides, peptides etc. subclasses: •esterases (lipases, phospholipases, ribonucleases, phosphatases) •glycosidases (e.g. sucrase, maltase, lactase, amylase) •proteinases, peptidases (pepsin, trypsin, cathepsins, caspases/apoptosis, dipeptidases, carboxypeptidases, aminopeptidases) •amidases (glutaminase, asparaginase) •ATPases (split anhydride bonds of ATP) ‹#› 20 Example: glucose 6-phosphatase glucose 6-P glucose ‹#› 21 Compare two antagonistic enzymes kinase phosphatase ‹#› 22 Glutaminase is amidase which catalyzes the deamidation of glutamine glutamate glutamine glutaminase ‹#› 23 aktin-myosin ATPase catalyzes the exergonic hydrolysis of phosphoanhydride bond in ATP ATP + H2O ® ADP + Pi + energy Example: muscle contraction myosine head exhibits ATPase activity, chemical energy of ATP is transformed into mechanical work (actin-myosin contraction) ‹#› 24 Examples of lysosomal hydrolases Hydrolase Bond hydrolyzed Glucosidase Galactosidase Hyaluronidase Arylsulfatase Lysozyme Cathepsin Collagenase Elastase Ribonuclease Lipase Phosphatase Ceramidase glycoside glycoside glycoside sulfoester glycoside peptide peptide peptide phosphodiester ester phosphoester amide ‹#› 25 Distinguish: lysozyme × lysosome •Lysozyme is enzyme •compound word, lyso (Greek lysis) + zyme (from enzyme) •hydrolase, glycosidase, cleaves β-1,4-glycoside bond in bacterial heteropolysaccharides, antiseptic defense •occurs in saliva, tears, and other body fluids • •Lysosome is intracellular digestion organelle •Greek compound word from lysis (to lyse) and soma (body) •typical for animal cells •acidic pH, contains many acidic hydrolases ‹#› 26 4 Lyases catalyze non-hydrolytic splitting or forming bonds C–C, C–O, C–N, C–S through removing or adding, respectively, a small molecule (H2O, CO2, NH3) Some frequent recommended names: • ammonia lyases (e.g. histidine ammonia lyase: histidine ® urocanate + NH3) • decarboxylases (amino acid ® amine + CO2) • aldolases (catalyze aldol cleavage and formation) • (de)hydratases (e.g. carbonate dehydratase: CO2 + H2O D H2CO3) Example: fumarate + H2O D L-malate Recommended name: fumarate hydratase Systematic name: (S)-malate hydro-lyase (fumarate-forming) ‹#› 27 5 Isomerases •catalyze intramolecular rearrangements of atoms •examples: •epimerases •racemases •mutases Example: UDP-glucose ® UDP-galactose Recommended name: UDP-glucose 4-epimerase Systematic name: UDP-glucose 4-epimerase ‹#› 28 6 Ligases •catalyze the formation of high-energy bonds C–C, C–O, C–N •in the reactions coupled with hydrolysis of ATP •Frequent recommended names: •carboxylases •synthetases •(e.g. glutamine synthetase: glutamate + ATP + NH3 ® glutamine + ADP + Pi) Example: pyruvate + CO2 + ATP + H2O ® oxaloacetate + ADP + Pi Recommended name: pyruvate carboxylase Systematic name: pyruvate:carbon-dioxide ligase (ADP-forming) ‹#› 29 Three enzymes dealing with phosphate Enzyme (Class) Reaction scheme / Reaction type Kinase (Transferase) substrate-OH + ATP ® substrate-O-P + ADP phosphorylation = transfer of phosphoryl PO32– from ATP to substrate Phosphatase (Hydrolase) substrate-O-P + H2O ® substrate-OH + Pi the hydrolysis of phosphoester bond Phosphorylase (Transferase) (glycogen)n + Pi ® (glycogen)n-1 + glucose 1-P inosine + Pi ® hypoxanthine + ribose 1-P phosphorolysis = the splitting of glycoside bond by phosphate = transfer of glucosyl to inorganic phosphate ! ‹#› 30 Distinguish: Three types of lysis (decomposition of substrate) Hydrolysis the decomposition of substrate by water, frequent in intestine: sucrose + H2O ® glucose + fructose (starch)n + H2O ® maltose + (starch)n-2 Phosphorolysis (see previous page) the cleavage of O/N-glycoside bond by phosphate: (glycogen)n + Pi ® (glycogen)n-1 + glucose 1-P Thiolysis the cleavage of C-C bond by sulfur atom of coenzyme A in β-oxidation of FA or ketone bodies catabolism RCH2COCH2CO-SCoA + CoA-SH ® RCH2CO-SCoA + CH3CO-SCoA CH3COCH2CO-SCoA + CoA-SH ® 2 CH3CO-SCoA ! ‹#› 31 Cofactors of enzymes •low-molecular non-protein compounds •many of them are derived from B-complex vitamins •many of them are nucleotides •transfer 2 H or e- (cooperate with oxidoreductases) •transfer groups (cooperate with transferases) •tightly (covalently) attached – prosthetic groups •loosely attached – coenzymes (cosubstrates) ‹#› 32 Three different components in enzyme reaction enzyme substrate cofactor D product cofactoraltered + + 1.substrate(s) 2.cofactor 3.enzyme catalyzes the whole process react to each other Notes: • one or two substrates may be involved (dehydrogenation × transamination) • substrate can be low / high molecular (hexokinase × protein kinase) • some reactions proceed without cofactor (hydrolysis, isomeration) • reaction can be reversible or irreversible (dehydrogenation × decarboxylation) ‹#› 33 Oxidized form Reduced form The function of cofactor NAD+ NADP+ FAD Dihydrobiopterin (BH2) Molybdopterinoxid Lipoate (-S-S-) Ubiquinone (Q) Heme-Fe3+ Non-heme-S-Fe3+ Glutathioneoxid (G-S-S-G) NADH+H+ NADPH+H+ FADH2 tetrahydrobiopterin (BH4) molybdopterinred dihydrolipoate (2 -SH) ubiquinol (QH2) heme-Fe2+ non-heme-S-Fe2+ glutathionered (GSH) NAD+ acceptor of 2H NADPH+H+ donor of 2H FAD acceptor of 2H BH4 donor of 2H electron transfer antioxidant / transfer of acyl transfer of 2 electrons + 2 H+ transfer of 1 electron transfer of 1 electron 2 GSH donor of 2H Cofactors of oxidoreductases ‹#› 34 NAD+ is the cofactor of dehydrogenases, derivative of nicotinamide (vitamin) •NAD+ is oxidant – takes off 2 H from substrate •one H adds as hydride ion (H-) into para-position of pyridinium cation of NAD+ •NAD+ + H- = NADH = equivalent of two electrons •the second H is released as proton (H+) and binds to enzyme molecule • 2 H = H– + H+ ‹#› 35 NAD+ (nicotinamide adenine dinucleotide) ribose diphosphate ribose N-glycosidic linkage N-glycosidic linkage addition of hydride anion anhydride adenine ‹#› 36 Redox pair of cofactor oxidized form NAD+ reduced form NADH aromatic ring aromaticity totally disturbed tetravalent nitrogen trivalent nitrogen positive charge on nitrogen electroneutral species high-energy compound ! ‹#› 37 Dehydrogenation by NAD+ •typical substrate groups: •primary alcohol -CH2-OH •secondary alcohol >CH-OH •secondary amine >CH-NH2 •double bond (C=O, C=N) is produced • ‹#› 38 NAD+ dehydrogenations form a double bond Substrate Product primary alcohol secondary alcohol aldehyde hydrate hemiacetal cyclic hemiacetal hydroxy acid amino acid aldehyde ketone carboxylic acid ester lactone oxo acid imino acid compare Med. Chem. II Appendix 3 ‹#› 39 Dehydrogenation of ethanol (alcohol dehydrogenase) ‹#› 40 Dehydrogenation of glutamate (glutamate dehydrogenase) glutamate 2-imino glutarate ‹#› 41 NAD+-dependent enzymes are called pyridine dehydrogenases •Citrate cycle • isocitrate dehydrogenase • 2-oxoglutarate dehydrogenase • malate dehydrogenase • •Glycolysis • glyceraldehyde 3-P dehydrogenase • lactate dehydrogenase • •Oxidation of ethanol • alcohol dehydrogenase • acetaldehyde dehydrogenase ‹#› 42 Reduced cofactor NADPH+H+ is hydrogenation agent •donor of 2 H in hydrogenations •cofactor of reducing syntheses (FA, cholesterol) •regeneration of glutathione (GSH) in erythrocytes •cofactor of hydroxylation reactions: • cholesterol ® ® bile acids • calciol ® ® calcitriol • xenobiotic ® hydroxylated xenobiotic •general scheme of hydroxylation: R-H + O2 + NADPH+H+ ® R-OH + H2O + NADP+ ‹#› 43 FAD is cofactor of flavin dehydrogenases, derivative of riboflavin (vitamin B2) •flavin adenine dinucleotide •dehydrogenation of -CH2-CH2- group •two H atoms are attached to two nitrogens of riboflavin (N-1 and N-10) •FAD + 2H ® FADH2 • • ‹#› 44 FAD (flavin adenine dinucleotide) vazba 2H adenine ribosa difosfát ribitol dimethylisoalloxazine ribose diphosphate the sites for accepting two H atoms ‹#› 45 Redox pair of cofactor oxidized form FAD reduced form FADH2 aromatic system aromaticity partially disturbed electroneutral species electroneutral species high-energy compound ! ‹#› 46 Dehydrogenation of succinate to fumarate (flavin dehydrogenase) ‹#› 47 Tetrahydrobiopterin (BH4) is a cofactor of hydroxylations •made in the body from GTP •donor of 2H •oxidized to dihydrobiopterin (BH2) guanine ‹#› 48 Redox pair of cofactor ‹#› 49 Hydroxylation of phenylalanine phenylalanine tyrosine ‹#› 50 Coenzyme Q (ubiquinone) •derivative of 1,4-benzoquinone •cyclic diketone, not aromatic •component of respiratory chain •gradually accepts electron and proton (2x) •reduced to semiubiquinone and ubiquinol ‹#› 51 Reversible reduction of ubiquinone R = long polyisoprenoid chain Þ lipophilic character electron (e-) and proton (H+) have different origin: electron comes from reduced cofactors, H+ from matrix of mitochondria (non-aromatic cycl. diketone) (aromatic ring + radical) (diphenol) ubiquinone semiubiquinone ubiquinol Q D •QH D QH2 ‹#› 52 Heme of various cytochromes •transfers just 1 electron •cytochromes are hemoproteins •components of respiratory chain or other heme enzymes (cyt P-450) •reversible redox reaction: Fe2+ D Fe3+ ‹#› 53 Non-heme iron (Fe2S2 cluster) transfers electron in R.CH. just one iron cation changes oxidation number oxidized state reduced state ‹#› 54 Xanthine oxidase catalyzes the oxygenation of purine bases (catabolism) hypoxanthine ¾® xanthine ¾® uric acid Molybdopterin (formula in Seminars) side product: H2O2 ‹#› 55 Sulfite oxidase: sulfate is catabolite from cysteine cysteine HSO3- + H2O ® SO42- + 3 H+ + 2 e- plasma urine acidify plasma urine reduce Mo Molybdopterin ‹#› 56 Redox pair lipoate/dihydrolipoate is antioxidant system. It is also involved in the acyl transfer (see later) oxidized form – lipoate (cyclic disulfide 1,2-dithiolane) reduced form - dihydrolipoate - 2H one S atom transfers acyl in oxidative decarboxylation of pyruvate / 2-oxoglutarate ‹#› 57 Glutathione (GSH) •tripeptide •γ-glutamyl-cysteinyl-glycine •cofactor of glutathione peroxidase (contains selenocysteine) •reduces H2O2 to water •2 G-SH + H-O-O-H ® G-S-S-G + 2 H2O • • Remember: The -SH compounds have generally reducing properties. ‹#› 58 Dehydrogenation of two GSH molecules ‹#› 59 Vitamin Cofactor Transferred group Pyridoxin (Made in body) (Made in body) Biotin Pantothenic acid (Made in body) (Methionine) Folate Cyanocobalamin Thiamin pyridoxal phosphate ATP PAPS carboxybiotin CoA-SH dihydrolipoate SAM tetrahydrofolate methylcobalamin thiamin diphosphate -NH2 (transamination) -PO32- (phosphoryl) -SO32- CO2 acyl acyl -CH3 C1 groups -CH3 residue of oxo acid Vitamins and cofactors of transferases ‹#› 60 Pyridoxal phosphate is the cofactor of transamination and decarboxylation of AA aldimine (Schiff base) - H2O transamination decarboxylation ‹#› 61 ATP is the cofactor of kinases (phosphorylation agent) N-glycoside bond ester anhydride ‹#› 62 Phosphorylation of substrate substrate kinase phosphorylated substrate CAUTION: creatine kinase (CK) phosphorylation on nitrogen (the bond N-P) ‹#› 63 PAPS is sulfation agent •3’-phosfoadenosine-5’-phosphosulfate •mixed anhydride of H2SO4 and H3PO4 •esterification of hydroxyl groups by sulfuric acid = sulfation •sulfated sphingoglycolipids •sulfated glycosaminoglycans (heparin, chondroitin sulfate, keratan sulfate) ‹#› 64 Carboxybiotin •cofactor of carboxylation reactions •carboxylation of biotin needs ATP carboxybiotin biotin ‹#› 65 Carboxybiotin is the cofactor of carboxylation reactions + pyruvate oxaloacetate pyruvate carboxylase ‹#› 66 Distinguish: Decarboxylation vs. Carboxylation Cofactor Decarboxylation (does not require energy) Thiamin-diP pyruvate ® acetyl-CoA + CO2 2-oxoglutarate ® succinyl-CoA + CO2 Pyridoxal-P amino acid ® amine + CO2 None acetoacetate ® acetone + CO2 (non-enzymatic, spontaneous) Cofactor Carboxylation (requires energy) Biotin pyruvate + CO2 + ATP ® oxaloacetate acetyl-CoA + CO2 + ATP ® malonyl-CoA propionyl-CoA + CO2 + ATP ® methylmalonyl-CoA ® succinyl-CoA carboxylations (ATP) in the catabolism of Val, Leu, Ile Phylloquinone (vitamin K) protein-glutamate + O2 + vit Kred + CO2 ® protein-γ-carboxyglutamate posttranslational carboxylation of glutamate ® hemostasis None Hb-NH2 + CO2 ® Hb-NH-COOH (unstable Hb-carbamate, spontaneous) ! ‹#› 67 Coenzyme A (CoA-SH) •transfers acyl •attached to sulfur atom •thioester bond •acyl-CoA is activated acyl •e.g. acetyl-CoA ‹#› 68 Coenzyme A ~ O O H C H 2 O P O O O O P O O O N N N N N H 2 H O P O O O C H 2 C HS C H 2 C H 2 H N O C C H 2 C H 2 H N O C C H C H 3 C H 3 cysteamine β-Alanine Pantoic acid pantothenic acid 3´-PhosphoADP acyl ‹#› 69 Lipoate (lipoamide) part of the 2-oxo acid dehydrogenase complex (see the following lectures) it is oxidant of a group carried by thiamine diphosphate (TDP), binds the resulting acyl as thioester and transfers the acyl to coenzyme A: S S CO–NH–Lys–Enzyme Lipoamide (oxidized form) S6-Acyldihydrolipoamide (reduced form) S H CO–NH–Lys–Enzyme S H S H CO–NH–Lys–Enzyme S R-CO – 2 H R1-CH–TDP OH CoA-SH R-CO–S-CoA Dihydrolipoamide (reduced form) H–TDP ‹#› 70 S-Adenosylmethionine (SAM) •„active methyl“, trivalent sulfur Þ sulfonium cation •cofactor of methylation reactions: • ethanolamine → choline (3× methylation) • guanidine acetate → creatine • noradrenaline → adrenaline ..... and many others •side product is homocysteine •remethylation of homocysteine needs methyl-FH4 + B12 cofactor (see Seminars) ‹#› 71 S-Adenosylmethionine (SAM) ‹#› 72 Folic acid is vitamin. In the body, it is hydrogenated to 5,6,7,8-tetrahydrofolate. amid 5 6 7 8 p-aminobenzoic acid glutamic acid amide pteridine transfer of 1C groups Tetrahydrofolate (FH4) is cofactor for the transfer of C1 groups ‹#› 73 C1 Groups transferred by FH4 Oxidation number of C Formula Name Metabolic Origin / Comment -III -CH3 methyl reduction of methylene-FH4 (from serine, glycine) methyl-FH4 cooperates with B12 cofactor in methylation -II -CH2- methylene catabolism of serine, glycine used in synthesis of dTMP ® DNA -I -CH= methenyl deamination of formimino-FH4 (from histidine) used in synthesis of purine bases +I -CH=O formyl catabolism of tryptophan ® formiate ® formyl used in synthesis of purine bases +I -CH=NH formimino catabolism of histidine compare scheme Seminars, p. 26 ‹#› 74 b12 B12 vitamin is cyano or hydroxocobalamin R = CN or OH corrin cycle hydroxocobalamin is used in the treatment of cyanide poisoning, it binds cyanide ions to nontoxic cyanocobalamin ‹#› 75 B12 cofactor is methyl or deoxyadenosylcobalamin, it is needed for two reactions in the body 1.homocysteine ® methionine methylation of homocysteine (regeneration of methionine) 2. 2.homocysteine ® ® propionyl-CoA ® ® succinyl-CoA 3. • B12 FH4 / B12 ‹#› 76 Compare: Four different cofactors of methylations Cofactor Origin of methyl Examples of methylation reactions SAM methionine ethanolamine ® choline guanidine acetate ® creatine noradrenaline ® adrenaline methylation of DNA (regulation of gene expression) methylation of bases in tRNA / mRNA (guanine-N7 = cap) inactivation of catecholamines (COMT): • dopamine ® methoxytyramine • noradrenaline ® normetanephrine • adrenaline ® metanephrine methylation of xenobiotics (II. phase - conjugation) methyl-FH4 methylene-FH4 homocysteine ® methionine methyl-B12 methyl-FH4 methylene-FH4 serine, glycine dUMP ® dTMP dUMP + methylene-H4F ® dTMP + H2F (thymidylate synthase) SAM = S-adenosylmethionine, FH4 = tetrahydrofolate, COMT = catechol O-methyltransferase ‹#› 77 Thiamin is vitamin B1 Thiamin diphosphate (TDP) is cofactor •Oxidative decarboxylation of some 2-oxo acids •pyruvate ® acetyl-CoA •2-oxoglutarate ® succinyl-CoA (citrate cycle) •2-oxo acids in the catabolism of branched amino acids (Val, Leu, Ile) • • • • • •Transketolase reactions in pentose cycle •ribose-5-P + xylulose-5-P D glyceraldehyde-3-P + sedoheptulose-7-P •xylulose-5-P + erythrose-4-P D fructose-6-P + glyceraldehyde-3-P • transfer to dihydrolipoate and CoA ‹#› 78 Thiamin diphosphate (TDP) is cofactor in the oxidative decarboxylation of pyruvate glucose ® pyruvate ® acetyl-CoA CAC TDP attachment of pyruvate and its decarboxylation ‹#› 79 In human body, a number of non-enzymatic reactions proceeds •decarboxylation of acetoacetate ® acetone •catabolism of creatine ® creatinine (dehydration + cyclization) •glycation / carbamylation / nitrosylation / nitration of proteins •the reactions of reactive oxygen species (e.g. lipoperoxidation) •spontaneous oxidation of hemoproteins (hemoglobin ® methemoglobin) •spontaneous oxidation of urobilinogens to urobilins (large intestine) •condensation of amines with carbonyl compounds to heterocyclic derivatives dopamine + pyruvate ® salsolinol (neurotoxin ?) • tryptamine + pyruvate ® harmane • dopamine + dihydroxyphenylacetaldehyde ® tetrahydropapaveroline •binding ligands to proteins: • bilirubin + albumin ® bilirubin-albumin complex • CO + hemoglobin ® carbonylhemoglobin •the interactions of macromolecules: • antigen + antibody ® immuno complex