1. Enzymes I  Department of Biochemistry, FM MU, 2011 (J.D.) General features , c ofa ctors

9. Hydrogenation of pyruvate When pyruvate is hydrogenated without enzyme ( in vitro ), the reaction product is the racemic mixture of D-lactate and L-lactate : In the same reaction catalyzed by lactate dehydrogenase (in the presence of NADH +H + ), pyruvate is reduced stereospecifically to L-lactate only: 1. non-chiral substrate  chiral product L-Lactate C COOH CH 3 H H O H C C CH 3 O O HO Enzyme L-Lactate D-Lactate

10. Non- enzym atic 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

11. Enzymatic hydration of fumarate (citrate cycle) in vivo just one enantiomer (L-malate) is produced reagent substrate 1. non-chiral substrate  chiral product enzyme

12. Hydrogenation of D-fructose in vitro gives two epimer s 2 H + D-fructose D-glucitol D-mannitol reaction site is planar i n vivo : enzym atic reaction gives just one product (D-glucitol) 1. non-chiral substrate  chiral product

13. Chiral substrates /signal molecules are bound to the stereospecific enzymes /receptors at three sites: Enzymes or receptors recognize only one enantiomer If the reactant of an enzymatic reaction is a chiral compound, only one of the two enantiomers is recognized as the specific substrate. 2. chiral substrate  product see also MCH II, p. 13 R R X x p roper enantiomer n ot-fitting enantiomer

14. 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/

16. Classification of enzymes: six classes according to reaction type (Each class comprises other subclasses) General scheme of reaction Enzyme class A + B + ATP  A-B + ADP + P i 6. Ligases (synthetases) A-B-C  A-C-B 5. Isomerases A-B  A + B (reverse reaction: synthases) 4. Lyases A-B + H 2 O  A-H + B-OH 3. Hydrolases A-B + C  A + C-B 2. Transferases A red + B ox  A ox + B red 1. Oxidoreductases

19. Example: P hosphor ylation of glucose glucose glucose 6-phosphate gluco kinase

21. Example: Glucose 6-phosphatase Glc 6-P Glc

22. Compare two antagonistic enzymes kinase phosphatase

23. Glutaminase is amidase which catalyzes the deamidation of glutamine glutam ate glutamin e glutamin ase

24. ATPase catalyzes the exergonic hydrolysis of phosphoanhydride bond in ATP ATP + H 2 O  ADP + P i + energ y Example: muscle contraction myosine head exhibits ATPase activity, chemical energy of ATP is transformed into mechanical work (= actin-myosin contraction)

25. Examples of lysosomal h ydrolas es gly c oside gly c oside gly c oside sulfoester gly c oside peptide peptide peptide phosphodiester ester phosphoester amide Glu c osidas e Gala c tosidas e Hyaluronidas e Arylsulfatas e Lysozym e C athepsin Collagenase Elastase Ribonuclease Lipase Phosphatase Ceramidase Bond hydrolyzed Hydrolas e

30. T hree enzym es have something to do with ph os phate ! (gly c ogen) n + P i  (gly c ogen) n-1 + glu c os e 1 -P inosine + P i  hypoxanthine + ribose 1-P ph os ph orol ysis = the splitting of gly c osid e bond by ph os phate = transfer of glu c osyl to in organic ph os phate Ph os ph orylas e (Transferas e ) substr ate -O-P + H 2 O  substr ate -OH + P i the h ydrol ysis of ph os ph oester bond Ph os ph atas e (Hydrolas e ) substr ate -OH + ATP  substr ate -O-P + ADP ph os ph oryla tion = transfer of ph os ph oryl PO 3 2– from ATP to substr ate Kinas e (Transferas e ) Reaction s ch eme / Reaction type Enzym e ( Class )

31. Distinguish : Three types of lysis (decomposition of substrate) ! the cleavage of C-C bond by s ulfur atom of coenzyme A in β -oxida tion of FA or keto ne bodies catabolism RCH 2 COCH 2 CO-SCoA + CoA-SH  RCH 2 CO-SCoA + CH 3 CO-SCoA Thiol ysis the cleavage of O / N -gly c osid e bond by ph os phate : (gly c ogen) n + P i  (gly c ogen) n-1 + glu c os e 1-P Ph os ph orol ysis (see previous page) the decomposition of substr ate by water , frequent in intestine: sucrose + H 2 O  glu c os e + fru c tos e ( starch ) n + H 2 O  maltos e + ( starch ) n-2 Hydrol ysis

34. Cofactors of oxidoreductases NAD + acceptor of 2H NADPH+H + donor of 2H FAD acceptor of 2H BH 4 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 The function of cofactor NADH+H + NADPH+H + FADH 2 tetrahydrobiopterin (BH 4 ) molybdopterin red dihydrolipoate (2 -SH) ubiquinol (QH 2 ) heme-Fe 2+ non-heme-S-Fe 2+ glutathione red (GSH) NAD + NADP + FAD Dihydrobiopterin (BH 2 ) Molybdopterin oxid Lipoate (-S-S-) Ubiquinone (Q) Heme-Fe 3+ Non-heme-S-Fe 3+ Glutathione oxid (G-S-S-G) Reduced form Oxidized form

36. NAD + ( n icotinamide a denine d inucleotide) ribose diphosphate ribose N-glycosidic linkage N-glycosidic linkage addition of hydride anion anhydride adenine

37. Redox p air of co fa c tor oxidized form NAD + reduced form NADH aromatic ring aromaticity totally disturbed tetravalent nitrogen trivalent nitrogen positive charge on nitrogen electroneutral species high-energy compound !

39. NAD + dehydrogena tions form a double bond compare Med. Chem. II Appendix 3 aldehyd e keton e c arboxyl ic acid ester la c ton e o xo acid i mino acid prim ary al c ohol se condary al c ohol a ldehyd e hydr ate hemi acetal cy clic hemiacetal h ydroxy acid a mino acid Produ c t Substr ate

40. Dehydrogenation of ethanol (alcohol dehydrogenase)

41. Dehydrogenation of glutamate (glutamate dehydrogenase) glutamate 2-imino glutarate

45. FAD ( f lavin a denine d inucleotide) vazba 2H adenine ribosa difosfát ribitol dimethylisoalloxazine ribose diphosphate the sites for accepting two H atoms

46. Redox p air of c ofa ctor oxidized form FAD reduced form FADH 2 aromatic system aromaticity partially disturbed electroneutral species electroneutral species high-energy compound !

47. Dehydrogenation of succinate to fumar ate (flavin dehydrogenas e )

49. Redox pair of cofactor

50. Hydroxyla tion of ph enylalanin e phenylalanine tyrosine

52. Reversible reduction of ubiquinone R = long polyisoprenoid chain  lipophilic character electron (e - ) and proton (H + ) have different origin: electron comes from red. c ofa c tor s (= nutrients ) , H + from matrix of mitochondrion ( n on-aromatic diketone) ( arom atic ring + radi cal) ( di ph enol ) ubiquinone semiubiquinone ubiquinol Q  • QH  QH 2

54. Non-heme iron (Fe 2 S 2 cluster) transfers electron in R.CH. just one iron cation changes oxidation number oxidized state reduced state

55. Xanthine oxidase catalyzes the oxygenation of purine bases (catabolism) hypoxanthine  xanthine  uric acid Molybdopterin (formula in Seminars) side product: H 2 O 2

56. Sulfite oxidase : sulfate is catabolite from cysteine cysteine HSO 3 - + H 2 O  SO 4 2- + 3 H + + 2 e - pla s ma urine acidify plasma urine reduce Mo (see Seminars, p. 46 ) Molybdopterin

57. 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

59. Dehydrogenation of two GSH molecules

60. Vitamins and cofactors of transferases -NH 2 (transamination) -PO 3 2- (phosphoryl) -SO 3 2- CO 2 acyl acyl -CH 3 C 1 groups -CH 3 r esidue of oxo acid Transferred group pyridoxal phosphate ATP PAPS carboxybiotin CoA-SH dihydrolipoate SAM tetrahydrofolate methylcobalamin t hiamin diphosphate Pyridoxin (Made in body) (Made in body) Biotin Pantothenic acid (Made in body) ( Methionin e) Folate Cyanocobalamin Thiamin Cofactor Vitamin

61. Pyridoxal phosphate is the cofactor of transamination and decarboxylation of AA aldimine (Schiff base) - H 2 O transamination decarboxylation

62. Two phases of transamina tion Schiff base amino acid oxo acid glutamate 2-oxoglutarate blue colour indicates the pathway of nitrogen

63. ATP is the cofactor of kinases (phosphorylation agent) N-glycoside bond ester anhydride

64. Phosphorylation of substrate substrate kinase phosphorylated substrate CAUTION: creatine kinase (CK) phosphorylation on nitrogen (the bond N-P)

67. Carboxybiotin is the cofactor of carboxylation reactions + pyruvate oxaloacetate pyruvate carboxylase

68. Distinguish: Decarboxylation vs. Carboxylation ! protein-glutamate + O 2 + vit K red + CO 2  protein- γ -carboxyglutamate posttranslational carboxylation of glutamate  hemostasis Phylloquinone (vitamin K) Hb-NH 2 + CO 2  Hb-NH-COOH (unstable Hb-carbamate, spontaneous) None pyruvate + CO 2 + ATP  oxaloacetate acetyl-CoA + CO 2 + ATP  malonyl-CoA propionyl-CoA + CO 2 + ATP  methylmalonyl-CoA  succinyl-CoA carboxylations (ATP) in the catabolism of Val, Leu, Ile Biotin Carboxylation (requires energy) Cofactor acetoacetate  acetone + CO 2 (non-enzymatic, spontaneous) None amino acid  amine + CO 2 Pyridoxal-P pyruvate  acetyl-CoA + CO 2 2-oxoglutarate  succinyl-CoA + CO 2 Thiamin-diP Decarboxylation (does not require energy) Cofactor

70. Coenzyme A acyl ~ 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

71. Lipo ate ( lipoamide ) part of the 2-oxo acid dehydrogenase complex (see the following lectures) it is oxidant of a group carried by thiamine d iphosphate (TDP ), binds the resulting acyl as thioester and transfers the acyl to coenzyme A: S S CO– NH –L ys–Enzyme Lipoamide ( oxidized form) S 6 -Acyldihydrolipoamide (reduced form) S H CO– NH – Lys–Enzyme S H S H CO– NH –L ys–Enzyme S R-CO – 2 H R 1 -C H – TDP O H CoA-S H R-CO–S-CoA Dihydrolipoamide ( reduced form) H –TDP

73. S -Adenosylmethionine (SAM)

74. Folic acid is vitamin. In the body, it is hydrogen ated to 5,6,7,8-tetrahydrofol ate . Tetrahydrofol ate (FH 4 ) is c ofa c tor for the transfer of C 1 groups amid 5 6 7 8 p-aminobenzoic acid glutamic acid amide pteridine transfer of 1C groups

75. C 1 Groups transferred by FH 4 compare scheme Seminars, p. 26 catabolism of histidine formimino -CH=NH +I catabolism of tryptophan  formiate  formyl synthesis of purine bases formyl -CH=O +I deamination of formimino-FH 4 (from histidine) synthesis of purine bases methenyl -CH= -I catabolism of serine, glycine used in synthesis of dTMP  DNA methylene -CH 2 - -II reduction of methylene-FH 4 (from serine, glycine) methyl-FH 4 cooperates with B 12 cofactor in methylation methyl -CH 3 -III Metabolic Origin / Comment Name Formula Oxidation number of C

76. B 12 vitamin is cyano or hydroxo cobalamin R = CN or OH c o r rin cycle hydroxo c obalamin is used in the treatment of cyanide poisoning - binds cyanide ions to non-toxic cyanocobalamin

80. Thiamin di phosphate (TDP) is the c ofa c tor of the oxida tive de c arboxyla tion of pyruv ate glu c os e  pyruv ate  acetyl-CoA C A C TDP attachment of pyruv ate and its de c arboxyla tion