Cofactors and Lipids

1. Cofactors and Lipids Andy HowardBiochemistry Lectures, Spring 201926 February 2019

2. Cofactors and Lipids • Many enzymes require capabilities available only in cofactors • Lipids are energy-storage molecules and are key components of membranes Cofactors and Lipids

3. Hemoglobin BPG Mutants Cofactors Metal ions Cosubstrates Prosthetic groups Lipids Fatty acids Triacylglycerols Phospholipids Sphingolipids Isoprenoids Steroids Topics for today Cofactors and Lipids

4. Another allosteric effector • 2,3-bisphosphoglycerate:heterotropic allosteric effector • Fairly prevalent in erythrocytes (4.5 mM); roughly equal to [Hb] • Hb tetramer has one BPG binding site • BPG effectively crosslinks the 2  chains • It only fits in T (deoxy) form! Cofactors and Lipids

5. BPG and physiology • pO2 is too high (40 Torr) for efficient release of O2 in many cells in absence of BPG • With BPG around, T-state is stabilized enough to facilitate O2 release • Big animals (e.g. sheep) have lower O2 affinity but their Hb is less influenced by BPG Cofactors and Lipids

6. Fetal hemoglobin • Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG • Fetal Hb is 22; doesn’t bind BPG as much as . • That helps ensure that plenty of O2 gets from mother to fetus across the placenta Cofactors and Lipids

7. Sickle-cell anemia • Genetic disorder: Hb residue 6 mutated from glu to val. This variant is called HbS. • Results in intermolecular interaction between neighboring Hb tetramers that can cause chainlike polymerization • Polymerized hemoglobin will partially fall out of solution and tug on the erythrocyte structure, resulting in misshapen (sickle-shaped) cells • Oxygen affinity is lower because of insolubility Cofactors and Lipids

8. Why has this mutation survived? • Homozygotes don’t generallysurvive to produce progeny;but heterozygotes do • Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled Deoxy HbS2.05 ÅPDB 2HBS Cofactors and Lipids

9. Heterozygotes and malaria BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem Cofactors and Lipids

10. How is sickling related to malaria? • Malaria parasite (Plasmodium spp.) infects erythrocytes • They’re unable to infect sickled cells • So a partially affected cell might survive the infection better than a non-sickled cell Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389 Cofactors and Lipids

11. Malaria and the tropics Still some argument about all of this Note that most tropical environments have plenty of oxygen around (not a lot of malaria at 2000 meters elevation) Cofactors and Lipids

12. Other hemoglobin mutants • Because it’s easy to get human blood, dozens of hemoglobin mutants have been characterized • Many are asymptomatic • Some have moderate to severe effects on oxygen carrying capacity or erythrocyte physiology Cofactors and Lipids

13. Cofactors and Vitamins Many enzymes require chemical functionalities unavailable from amino acids by themselves; they therefore employ cofactors Cofactors and Lipids

14. Family tree of cofactors • Cofactors, coenzymes, essential ions, cosubstrates, prosthetic groups: Cofactors(apoenzyme + cofactor  holoenzyme) Essential ions Coenzymes Activator ions(loosely bound) Ions inmetalloenzymes Prosthetic groups(tightly bound) Cosubstrates(loosely bound) Cofactors and Lipids

15. Metal-activated enzymes • Absolute requirements for mobile ions • Often require K+, Ca2+, Mg2+ • Example: Kinases: Mg-ATP complex • Metalloenzymes: firmly bound metal ions in active site • Usually divalent or more • Sometimes 1e- redox changes in metal Cofactors and Lipids

16. Coenzymes • Organic moieties that enable enzymes to perform their function: they supply functionalities not available from amino acid side chains • Those functions typically involve a change in the state of the coenzyme: • it might change oxidation state • lose a phosphate group • break a disulfide Cofactors and Lipids

17. Two kinds of coenzymes • Cosubstrates • Enter reaction, get altered, leave • Repeated recycling within cell or organelle • Prosthetic groups • Remain bound to enzyme throughout • Change during one phase of reaction, eventually get restored to starting state Cofactors and Lipids

18. Coenzyme precursors • 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+ • Some coenzyme precursors are, in fact, lipidic Cofactors and Lipids

19. 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 Cofactors and Lipids

20. Major cosubstrates • Facilitate group transfers, mostly small groups • Oxidation-reduction participants Cosubstrate Source Function ATP Transfer P,Nucleotide S-adenosylMet Methyl transfer UDP-glucose Glycosyl transfer NAD,NADP Niacin 2-electron redox Coenzyme A Pantothenate Acyl transfer Tetrahydrofolate Folate 1Carbon transfer Ubiquinone Lipid-soluble e- carrier Cofactors and Lipids

21. Prosthetic groups • Transfer of larger groups • One- or two-electron redox changes Prosth.gp. Source Function FMN, FAD Riboflavin 1e- and 2e- redox transfers TPP Thiamine 2-Carbon transfers with C=O PLP Pyridoxine Amino acid group transfers Biotin Biotin Carboxylation, COO- transfer Adenosyl- Cobalamin Intramolec. rearrangements cobalamin MeCobal. Cobalamin Methyl-group transfers Lipoamide Transfer from TPP Retinal Vitamin A Vision Vitamin K Vitamin K Carboxylation of glu residues Cofactors and Lipids

22. Which structures must we memorize? • ATP, NAD, NADP • … but that’s not very hard: • You’ll memorize adenine and ribose anyway • Phosphate is easy • So the only new moiety you need to memorize is nicotinamide nicotinamide Cofactors and Lipids

23. Adenosine triphosphate • Synthesizable in liver • Participates in phosphoryl-group transfer in kinases • Source of other coenzymes • Not derived from a vitamin; we’ll see how it’s synthesized later Cofactors and Lipids

24. S-adenosylmethionine • Made from methionine and adenosine • Sulfonium group is highly reactive: can donate methyl groups Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University Cofactors and Lipids

25. UDP-glucose • Most common donor of glucose • Formed via:Glucose-1P + UTPUDP-glucose + PPi • Reaction driven to right by PPi hydrolysis Cofactors and Lipids

26. 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 Cofactors and Lipids

27. 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 Cofactors and Lipids

28. Differences between NAD(H) and NADP(H) • 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 Cofactors and Lipids

29. Typical NAD+ NADH and NADPH  NADP+ reactions Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products NAD+ + R1R2CHOH  NADH + R1R2C=O + H+ R–CH=CH–CO-S-ACP + NADPH + H+R–CH2CH2CO-S-ACP + NADP+ [NAD+] >> [NADH] but [NADPH] >> [NADP+] Cofactors and Lipids

30. How do we get back to the starting point? • NADH often oxidized back to NAD+:part of the electron-transport chain • Photosynthesis & PPP make NADPH • Imbalances can be addressed via: • NAD Kinase (S.Kawai et al (2005),J.Biol.Chem.280:39200) • NADP phosphatase Listeria NADK32kDa monomerEC 2.7.1.23 PDB 2I2C, 1.9Å Cofactors and Lipids

31. Coenzyme A • Reactive portion is free sulfhydryl at one end of the molecule • Can form thioester with acetate, etc. • Pantoate + b-alanine = pantothenate Cofactors and Lipids

32. Structure of coenzyme A ADP with 3’-phosphate group β-alanine 2-mercapto-ethylamine  pantoate  KlebsiellaPanthothenate kinase78 kDa dimerEC 2.7.1.33PDB 4NE2, 1.9Å β-alanine + pantoate =pantothenate Cofactors and Lipids

33. Tetrahydrofolate • Primary donor of one-carbon units(formyl, methylene, methyl) • Supplies methyl group for thymidylate • Dihydrofolate reductase (DHFR):drug target • Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells • Trimethoprim as antibacterial:Bacterial DHFR differs from eukaryotic DHFR because bacteria derive DHF from other sources Cofactors and Lipids

34. THF structure & function Figure courtesy horticulture program, Purdue Cofactors and Lipids

35. THF derived from folic acid Folate is a vitamin for humans Deficiency gives rise to megaloblastic anemia and is linked to birth defects in babies born to folate-deficient mothers, including neural tube defects like spina bifida Cofactors and Lipids

36. Ubiquinone Also called coenzyme Q Contains quinone moiety that can undergo one- (or two-)electron reductions Phytol side-chain allows for embedding into membrane: many of its reactions occur in mitochondrial membrane (see ch. 14) Humans can make it: 10-gene system Cofactors and Lipids

37. Ubiquinone: role and relatives Stronger oxidizer than NAD+, FMN, FAD So it’s reduced by NADH, FADH2 Moves electrons from one side of a membrane to another: see chapter 14 Antioxidant: helps prevent lipid peroxidation in membranes Closely-related cofactors in plants, bacteria: menaquinone, plastoquinone Cofactors and Lipids

38. FAD and FMN riboflavin • Flavin group based on riboflavin • Alternate participants in redox reactions • Prosthetic groups: tightly but noncovalently bound to their enzymes • Protects against wasteful reoxidation of reduced forms • FADH2 is weaker reducing agent than NADH • These are capable of one-electron oxidations and reductions as well as 2e- redox Cofactors and Lipids

39. FAD and FMN structures • FAD has an AMP attached P to P Flavin adenine dinucleotide Flavin mononucleotide Cofactors and Lipids

40. FMN/FAD redox forms • Two-electron version: H+ + :H- transferred Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University Cofactors and Lipids

41. Thiamine Pyrophosphate 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:pKa drops from 15 in H2O to 6 in enzyme Cofactors and Lipids

42. TPP reactions pyrimidine thiazolium Diagram courtesy ofOklahoma State U.Biochemistry program Cofactors and Lipids

43. Pyridoxal phosphate • PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations • Based on pyridoxine, vitamin B6 • Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site Pyridoxal phosphate Cofactors and Lipids

44. PLP mechanisms First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine Cofactors and Lipids

45. PLP-dependent Transaminations • -amino acid1 + -ketoacid2-ketoacid1 + -amino acid2 • Example: • -amino acid1 = aspartate • -ketoacid2 = -ketoglutarate • -ketoacid1 = oxaloacetate • -amino acid2 = glutamate Sus aspartate aminotransferase94 kDa dimerEC 2.6.1.1PDB 5TOQ, 1.2Å Cofactors and Lipids

46. Significance of transaminations Many biosynthetic / degradative pathways for normal amino acids depend on these reactions Cofactors and Lipids

47. Biotin (vitamin B7) Biotin • Rarity: vitamin is the prosthetic group • Used in reactions that transfer carboxyl groups • … and in ATP-dependent carboxylations Rhizobium pyruvate carboxylase258 kDa heterodimer, EC 6.4.1.1PDB 2QF7, 2Å Cofactors and Lipids

48. Biotin reactivity • Covalently bound to active-site lysines to form species called biocytin • Pyruvate carboxylase is characteristic reaction: Cofactors and Lipids

49. Cobalamin and derivatives • Largest B vitamin; cobalt bound in core • Corrin ring: like heme but missing one carbon in ring structure • Involved in enzymatic rearrangements • Catabolism of odd-chain fatty acids • Methylation of homocysteine • Reductive dehalogenation Cofactors and Lipids

50. Adenosyl-cobalamin Reactive Co-C bond “missing” carbon Adenosyl donor, e.g. in methylmalonyl-CoA mutase Cofactors and Lipids