1. 11/11/2008Biochem: Cofactors,Vitamins Cofactors & Vitamins Andy Howard Introductory Biochemistry 11 November 2008

2. 11/11/2008Biochem: Cofactors,Vitamins Page 2 of 49 What we’ll discuss Coenzymes NAD and NADP FAD and FMN Coenzyme A TPP, PLP Biotin, THF Cobalamin Lipoamide Vitamins Why they exist Water-soluble vitamins Cofactor precursors Ascorbate Fat-Soluble Vitamins

3. 11/11/2008Biochem: Cofactors,Vitamins Page 3 of 49 Major cosubstrates (review) Facilitate group transfers, mostly small groups Oxidation-reduction participants CosubstrateSourceFunction ATPTransfer P,Nucleotide S-adenosylMetMethyl transfer UDP-glucoseGlycosyl transfer NAD,NADPNiacin2-electron redox Coenzyme APantothenateAcyl transfer TetrahydrofolateFolate1Carbon transfer UbiquinoneLipid-soluble e - carrier

4. 11/11/2008Biochem: Cofactors,Vitamins Page 4 of 49 Major prosthetic groups (rvw) Transfer of larger groups One- or two-electron redox changes Prosth.gp.SourceFunction FMN, FADRiboflavin1e - and 2e - redox transfers TPPThiamine2-Carbon transfers with C=O PLPPyridoxineAmino acid group transfers BiotinBiotinCarboxylation, COO - transfer Adenosyl-CobalaminIntramolec. rearrangements cobalamin MeCobal.CobalaminMethyl-group transfers LipoamideTransfer from TPP RetinalVitamin AVision Vitamin KVitamin KCarboxylation of glu residues

5. 11/11/2008Biochem: Cofactors,Vitamins Page 5 of 49 NAD + and NADP + Net charge isn’t really >0 ; the + is just a reminder that the nicotinamide ring is positively charged Most important cosubstrates in oxidation- reduction reactions in aerobic organisms Structure courtesy of Sergio Marchesini, U. Brescia

6. 11/11/2008Biochem: Cofactors,Vitamins Page 6 of 49 Differences between them The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety The functional difference is that NAD + is usually associated with catabolic reactions and NADP + is usually associated with anabolic reactions Therefore often NAD + and NADPH are reactants and NADH and NADP + are products

7. 11/11/2008Biochem: Cofactors,Vitamins Page 7 of 49 How do we get back to the starting point? NADH is often oxidized back to NAD + as part of the electron-transport chain Imbalances can be addressed via: NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) NADP phosphatase

8. 11/11/2008Biochem: Cofactors,Vitamins Page 8 of 49 iClicker quiz: question 1 What would you expect to be the phosphate donor in the NAD kinase reaction? (a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate

9. 11/11/2008Biochem: Cofactors,Vitamins Page 9 of 49 Reduced forms of NAD(P) Reduction occurs on the nicotinamide ring Ring is no longer net- positive Ring is still planar but the two hydrogens on the para carbon are not

10. 11/11/2008Biochem: Cofactors,Vitamins Page 10 of 49 FAD and FMN Flavin group based on riboflavin Alternate participants in redox reactions Prosthetic groups: tightly but noncovalently bound to their enzymes That protects against wasteful reoxidation of reduced forms FADH 2 is weaker reducing agent than NADH These are capable of one-electron oxidations and reductions

11. 11/11/2008Biochem: Cofactors,Vitamins Page 11 of 49 FAD and FMN structures FAD has an AMP attached P to P Structure courtesy Paisley University

12. 11/11/2008Biochem: Cofactors,Vitamins Page 12 of 49 Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University FMN/FAD redox forms Two-electron version: H + + :H - transferred

13. 11/11/2008Biochem: Cofactors,Vitamins Page 13 of 49 Coenzyme A Reactive portion is free sulfhydryl at one end of the molecule Can form thioester with acetate, etc. Pantoate +  -alanine = pantothenate Structure courtesy of MPB project, George Washington University (ADP-3’P) (Pantoate)  -alanine)  2-mercapto- ethylamine)

14. 11/11/2008Biochem: Cofactors,Vitamins Page 14 of 49 Thiamine Pyrophosphate Based on thiamine, vitamin B1 Many carboxylases and oxidative decarboxylases use this coenzyme So do transketolases (move 2 carbons at a time between sugars with keto groups) Thiazolium ring is reactive center: pK a drops from 15 in H 2 O to 6 in enzyme

15. 11/11/2008Biochem: Cofactors,Vitamins Page 15 of 49 TPP reactions Diagram courtesy of Oklahoma State U. Biochemistry program pyrimidine thiazolium

16. 11/11/2008Biochem: Cofactors,Vitamins Page 16 of 49 Pyridoxal phosphate PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations Carbonyl group of PLP bound as a Schiff base (imine) to  -amino group of lysine at active site First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine

17. 11/11/2008Biochem: Cofactors,Vitamins Page 17 of 49 Transaminations  -amino acid 1 +  -ketoacid 2   -ketoacid 1 +  -amino acid 2 Example:  -amino acid 1 = glutamate  -ketoacid 2 = pyruvate  -ketoacid 1 = a-ketoglutarate  -amino acid 2 = alanine Many biosynthetic / degradative pathways for normal amino acids depend on these reactions

18. 11/11/2008Biochem: Cofactors,Vitamins Page 18 of 49 Biotin Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl groups … and in ATP-dependent carboxylations

19. 11/11/2008Biochem: Cofactors,Vitamins Page 19 of 49 Biotin reactivity Covalently bound to active-site lysines to form species called biocytin Pyruvate carboxylase is characteristic reaction: Diagram courtesy University of Virginia Biochemistry

20. 11/11/2008Biochem: Cofactors,Vitamins Page 20 of 49 Tetrahydrofolate Primary donor of one-carbon units (formyl, methylene, methyl) Supplies methyl group for thymidylate Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells Trimethoprim as antibacterial: Bacterial DHFR is somewhat different from eucaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate

21. 11/11/2008Biochem: Cofactors,Vitamins Page 21 of 49 THF structure and function Figure courtesy horticulture program, Purdue

22. 11/11/2008Biochem: Cofactors,Vitamins Page 22 of 49 Cobalamin Largest B vitamin Corrin ring structure related to heme but missing one carbon in ring structure Cobalt bound in core of ring system Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation

23. 11/11/2008Biochem: Cofactors,Vitamins Page 23 of 49 Adenosyl- Cobalamin Diagram courtesy of Swiss Food News “Missing” carbon Reactive Co-C bond

24. 11/11/2008Biochem: Cofactors,Vitamins Page 24 of 49 Lipoamide Protein-bound form of lipoic acid Contains five-membered disulfide ring Covalently bound via amide to protein lysine sidechain Involved in swinging arm between active sites in multienzyme complexes Disulfides break periodically Example: pyruvate dehydrogenase complex

25. 11/11/2008Biochem: Cofactors,Vitamins Page 25 of 49 Lipoamide 2e - reduction Cf. Scheme 7.6: thioester starting point Fig. Courtesy Biochem and Biophysics program, Rensselaer

26. 11/11/2008Biochem: Cofactors,Vitamins Page 26 of 49 iClicker revisited Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate  aspartate +  -ketoglutarate? (a) ascorbate (b) PLP ( c) thiamine pyrophosphate (d) NAD (e) none of the above

27. 11/11/2008Biochem: Cofactors,Vitamins Page 27 of 49 Many cofactors are derived from vitamins We justify lumping these two topics together because many cofactors are vitamins or are metabolites of vitamins.

28. 11/11/2008Biochem: Cofactors,Vitamins Page 28 of 49 Vitamins: necessary micronutrients that cannot be synthesized internally What’s a vitamin for one organism is not for another Primates and some rodents are the only vertebrates that don’t synthesize ascorbate E.coli can make almost everything given energy and sources of atoms

29. 11/11/2008Biochem: Cofactors,Vitamins Page 29 of 49 Why wouldn’t organisms make everything? Complex metabolites require energy for synthesis Control of their synthesis is also metabolically expensive Cheaper in the long run to derive these nutrients from diet

30. 11/11/2008Biochem: Cofactors,Vitamins Page 30 of 49 Vitamins: broad classifications Water-soluble vitamins Coenzymes or coenzyme precursors Non-coenzymic metabolites Fat-soluble vitamins Antioxidants Other lipidic vitamins

31. 11/11/2008Biochem: Cofactors,Vitamins Page 31 of 49 Are all nutrients that we can’t synthesize considered vitamins? No: If it’s required in large quantities, it’s not a vitamin By convention, essential fatty acids like arachidonate aren’t considered vitamins

32. 11/11/2008Biochem: Cofactors,Vitamins Page 32 of 49 Coenzyme precursors We’ve already outlined the fact that most water-soluble coenzymes are derived from vitamins—typically B vitamins Typically the dietary form can be converted by a fairly short metabolic pathway into the coenzyme form, e.g. niacin + glutamine  nicotinamide + glutamate nicotinamide + ADP-ribose  NAD

33. 11/11/2008Biochem: Cofactors,Vitamins Page 33 of 49 The B vitamins All aqueous micronutrients Generally identified via pathologies associated with dietary deficiencies B1: thiamin (produces TPP) B2: riboflavin (produces FAD, FMN) B3: niacin (produces NAD, NADP) B5: pantothenate (produces Coenzyme A) B6: pyridoxamine (produces PLP) B9: folate: produces THF, THF derivatives B12: cobalamin (produces adenosylcobalamin, methylcobalamin)

34. 11/11/2008Biochem: Cofactors,Vitamins Page 34 of 49 Deficiency of niacin Pellagra: dermatitis, diarrhea, dementia Still found in some diets that are low in vitamin content Humans can actually synthesize nicotinamide from tryptophan; but that’s often in short supply too Image courtesy history.nih.gov

35. 11/11/2008Biochem: Cofactors,Vitamins Page 35 of 49 Deficiency of thiamine Beriberi: primary symptoms are in nervous system and musculature Polished rice is missing thiamine; rice hulls are rich in it Image courtesy answers.com

36. 11/11/2008Biochem: Cofactors,Vitamins Page 36 of 49 Deficiency of cobalamin Available sources of cobalamin are animal products Therefore vegans need to be careful to get cobalamin from supplements Symptoms of deficiency (pernicious anemia): weakness, fatigue, pallor, palpitations, dizziness Deficiency is common even in non-vegans: 5-40% of the population?

37. 11/11/2008Biochem: Cofactors,Vitamins Page 37 of 49 Ascorbate Vitamin in primates, some rodents Synthesizable in most other vertebrates Involved in collagen processing Reduced form acts as reducing agent during hydroxylation of collagen Deficiency gives rise to inadequate collagen - scurvy

38. 11/11/2008Biochem: Cofactors,Vitamins Page 38 of 49 PTM role of ascorbate (fig. 6.17) Proline + O 2 +  -ketoglutarate + ascorbate  4-hydroxyproline + succinate + CO 2 + dehydroascorbate This is a post-translational modification that occurs to prolines within collagen The hydroxylated prolines help stabilize the collagen triple helix Hydroxylysine found in collagen too

39. 11/11/2008Biochem: Cofactors,Vitamins Page 39 of 49 Dietary deficiency of ascorbate Primary sources of ascorbate are fruits, particularly citrus, and green vegetables Ascorbate deficiency’s first symptom involves collagen degradation, leading to scurvy Image courtesy U.Cincinnati Medical School

40. 11/11/2008Biochem: Cofactors,Vitamins Page 40 of 49 Scurvy in history Shortage of green vegetables in sailors’ diets meant scurvy was rampant on shipboard until the 18th century Success of English navy over French 1760-1800 was partly due to the introduction of limes in English sailors’ diets 50 years before the French caught on

41. 11/11/2008Biochem: Cofactors,Vitamins Page 41 of 49 Lipid vitamins Contain rings & long aliphatic sidechains At least one polar group in each Absorbed in intestine, carried via bile salts Hard to study Most are formally built from isoprene units, as are steroids

42. 11/11/2008Biochem: Cofactors,Vitamins Page 42 of 49 Vitamin A (retinol) 3 forms varying in terminal polar group Involved in signaling and receptors  -carotene is nonpolar dimer

43. 11/11/2008Biochem: Cofactors,Vitamins Page 43 of 49 Vitamin A deficiency Produces night blindness because the retina and cornea dry out Most common cause: nursing infants whose mothers have vitamin A deficiency in their diet

44. 11/11/2008Biochem: Cofactors,Vitamins Page 44 of 49 Vitamin D Several related forms Hormones involved in Ca 2+ regulation Figure courtesy Cyberlipid (cholecalciferol)

45. 11/11/2008Biochem: Cofactors,Vitamins Page 45 of 49 Vitamin D deficiency Rickets in children: Bone disease, restlessness, slow growth One form of vitamin D is actually synthesizable from cholesterol given adequate sunlight; Therefore rickets is most common in densely settled urban environments