1. 1 Vitamins, Allostery, and Sugars Andy Howard Introductory Biochemistry, Spring 2008 26 February 2008

2. Biochemistry: Vitamins; Carbohydrates I p. 2 of 58 Now we’ll study sugars! But first, we’ll finish discussing vitamins, and we’ll pick up a specific protein-related topic from chapter 5—namely, allostery

3. Biochemistry: Vitamins; Carbohydrates I p. 3 of 58 What we’ll discuss Vitamins Water-soluble Fat-Soluble Allostery Carbohydrates Sugar Concepts Monosaccharides Oligosaccharides Carbohydrates Glycosides Polysaccharides Starch & glycogen Cellulose and chitin Glycoconjugates Proteoglycans Peptidoglycans Glycoproteins

4. Biochemistry: Vitamins; Carbohydrates I p. 4 of 58 Vitamins: broad classifications Water-soluble vitamins Coenzymes or coenzyme precursors Non-coenzymic metabolites Fat-soluble vitamins Antioxidants Other lipidic vitamins

5. Biochemistry: Vitamins; Carbohydrates I p. 5 of 58 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

6. Biochemistry: Vitamins; Carbohydrates I p. 6 of 58 Ascorbate Vitamin in primates, some rodents Synthesizable in most other vertebrates Involved in collagen Reduced form acts as reducing agent during hydroxylation of collagen: proline  hydroxyproline, lysine  hydroxylysine Deficiency gives rise to inadequate collagen - scurvy

7. Biochemistry: Vitamins; Carbohydrates I p. 7 of 58 Vitamin A (retinol) 3 forms varying in terminal polar group Involved in signaling and receptors  -carotene is nonpolar dimer

8. Biochemistry: Vitamins; Carbohydrates I p. 8 of 58 Vitamin D Several related forms Hormones involved in Ca 2+ regulation Figure courtesy Cyberlipid

9. Biochemistry: Vitamins; Carbohydrates I p. 9 of 58 Vitamin E (  -tocopherol) Phenol can undergo 1e - oxidation to moderately stable free radical Antioxidant activity prevents damage to fatty acids in membranes Fig. Courtesy UIC pharmacy program phenol

10. Biochemistry: Vitamins; Carbohydrates I p. 10 of 58 Vitamin K (phylloquinone) Involved in synthesis of proteins involved in blood coagulation Reduced form involved as reducing agent in carboxylation reaction on glu sidechains Figure courtesy Cyberlipid

11. Biochemistry: Vitamins; Carbohydrates I p. 11 of 58 Allostery This is covered at the end of chapter 5, but somehow we missed it… here goes. Formal definition: alterations in protein function that occur when the structure changes upon binding of small molecules In practice: often the allosteric effector is the same species as the substrate

12. Biochemistry: Vitamins; Carbohydrates I p. 12 of 58 What it means for enzymes Non-enzymatic proteins can be allosteric: hemoglobin’s affinity for O 2 is influenced by the binding of O 2 to other subunits In enzymes: non-MM kinetics (often sigmoidal) when the allosteric activator is also the substrate v0v0 [S]

13. Biochemistry: Vitamins; Carbohydrates I p. 13 of 58 R and T states Enzyme with multiple substrate binding sites is in T (“tense”) state in absence of substrate Binding of substrate moves enzyme into R (“relaxed”) state where its affinity for substrate at other sites is higher Velocity can then rise rapidly as function of [S] Once all the enzyme is converted to R state, ordinary hyperbolic kinetics take over

14. Biochemistry: Vitamins; Carbohydrates I p. 14 of 58 Other effectors can influence R  T transitions Post-translational covalent modifiers often influence R  T equilibrium Phosphorylation can stabilize either the R or T state Binding of downstream products can inhibit T  R transition Binding of alternative metabolites can stabilize R state

15. Biochemistry: Vitamins; Carbohydrates I p. 15 of 58 Why does that make sense? Suppose reactions are: (E) A  B  C  D Binding D to enzyme E that converts A to B will destabilize R state, limiting conversion of A to B and (ultimately) reducing / stabilizing [D]: homeostasis!

16. Biochemistry: Vitamins; Carbohydrates I p. 16 of 58 Alternative pathways Often one metabolite has two possible fates: B  C  D A H  I  J If we have a lot of J around, it will bind to the enzyme that converts A to B and activate it; that will balance D with J!

17. Biochemistry: Vitamins; Carbohydrates I p. 17 of 58 Carbohydrates These are polyhydroxylated aldehydes and ketones, many of which can exist in cyclic forms General monomeric formula (CH 2 O) m, 3 < m < 9 With one exception (dihydroxyacetone) they contain chiral centers Highly soluble Can be oligomerized and polymerized Most abundant organic molecules on the planet

18. Biochemistry: Vitamins; Carbohydrates I p. 18 of 58 How do we measure solubility for very soluble compounds? (Note: this is not a serious chemical topic: it’s an example of how statistics can be abused…) The assertion is that, with highly soluble compounds like sugars, it’s difficult to use conventional approaches to compare their solubilities The suggestion is that we might use the amount of time it takes to dissolve (for example) 50g of solute in 100mL of cold water: if it’s fast, the solute is more soluble than if it’s slow.

19. Biochemistry: Vitamins; Carbohydrates I p. 19 of 58 Solubility measured by dissolution time Assertion: more polar groups means shorter dissolution time for a given class of compounds # of polar groups Time required for dissolution 1 2 3 4 5 6

20. Biochemistry: Vitamins; Carbohydrates I p. 20 of 58 What if we extrapolate to n=6? We get a negative dissolution time! That is, the solid goes into solution 6 seconds before we put it in the water! This causes serious psychological problems (what if I change my mind?) and philosophical problems (is this pre-ordained?) # of polar groups Time required for dissolution 1 2 3 4 5 6 Observed points Extrapolated point

21. Biochemistry: Vitamins; Carbohydrates I p. 21 of 58 Whose idea is this? Isaac Asimov, that’s who! “The endochronic properties of resublimated thiotimolene”: Astounding Science Fiction, ~1948 His point: extrapolations and other misuses of statistics are dangerous Benjamin Disraeli (popularized by Mark Twain): There are three kinds of untruth: lies, damn lies, and statistics.

22. Biochemistry: Vitamins; Carbohydrates I p. 22 of 58 Aldoses & ketoses If the carbonyl moiety is at the end carbon (conventionally counted as 1), it’s an aldose If carbonyl is one carbon away (counted as 2), it’s a ketose If it’s two or more carbons from the end of the chain, it’s not a sugar

23. Biochemistry: Vitamins; Carbohydrates I p. 23 of 58 Simplest monosaccharides Glyceraldehyde and dihydroxyacetone Only glyceraldehyde is chiral: D-enantiomer is more plentiful in biosphere All longer sugars can be regarded as being built up by adding -(CHOH) m-1 to either glyceraldehyde or dihydroxyacetone, just below C2

24. Biochemistry: Vitamins; Carbohydrates I p. 24 of 58 How many aldoses are there? Every -(CHOH) in the interior offers one chiral center An m-carbon aldose has (m-2) internal -(CHOH) groups Therefore: 2 m-2 aldoses of length m For m=3, that’s 2 1 =2; for m=6, it’s 2 4 =16.

25. Biochemistry: Vitamins; Carbohydrates I p. 25 of 58 How many ketoses are there? Every -(CHOH) in the interior offers one chiral center An m-carbon ketose has (m-3) internal -(CHOH) groups Therefore: 2 m-3 ketoses of length m For m=3, that’s 2 0 = 1; for m=6, that’s 2 3 =8.

26. Biochemistry: Vitamins; Carbohydrates I p. 26 of 58 Review: stereochemical nomenclature Stereoisomers: compounds with identical covalent bonding apart from chiral connectivity Enantiomers: compounds for which the opposite chirality applies at all chiral centers Epimers: compounds that differ in chirality at exactly one chiral center One chiral center: enantiomers are epimers. > 1 chiral center: enantiomers are not epimers.

27. Biochemistry: Vitamins; Carbohydrates I p. 27 of 58 Example: 2 chiral centers Chiral centers u,v; compounds A,B,C,D Compound Stereo @ u Stereo @ v Enantio- morph of Epimer of A++DB,C B+-CA,D C-+B D--AB,C

28. Biochemistry: Vitamins; Carbohydrates I p. 28 of 58 Properties Enantiomers have identical physical properties (MP,BP, solubility, surface tension…) except when they interact with other chiral molecules Stereoisomers that aren’t enantiomers can have different properties; therefore, they’re often given different names

29. Biochemistry: Vitamins; Carbohydrates I p. 29 of 58 Sugar nomenclature All sugars with m≤7 have specific names apart from their enantiomeric (L or D) designation, e.g. D-glucose, L-ribose.

30. Biochemistry: Vitamins; Carbohydrates I p. 30 of 58 Fischer projections Convention for drawing open- chain monosaccharides If the hydroxyl comes off counterclockwise relative to the previous carbon, we draw it to the left; Clockwise to the right. Emil Fischer

31. Biochemistry: Vitamins; Carbohydrates I p. 31 of 58 Cyclic sugars Sugars with at least four carbons can readily interconvert between the open- chain forms we have drawn and five- membered(furanose) or six-membered (pyranose) ring forms in which the carbonyl oxygen becomes part of the ring There are no C=O bonds in the ring forms

32. Biochemistry: Vitamins; Carbohydrates I p. 32 of 58 Furanoses Formally derived from structure of furan Hydroxyls hang off of the ring; stereochemistry preserved there Extra carbons come off at 2 and 5 positions 3 2 1 4 5 furan

33. Biochemistry: Vitamins; Carbohydrates I p. 33 of 58 Pyranoses Formally derived from structure of pyran Hydroxyls hang off of the ring; stereochemistry preserved there Extra carbons come off at 2 and 6 positions 3 2 4 5 1 6 pyran

34. Biochemistry: Vitamins; Carbohydrates I p. 34 of 58 How do we cyclize a sugar? Formation of an internal hemiacetal or hemiketal (see a few slides from here) by conversion of the carbonyl oxygen to a ring oxygen Not a net oxidation or reduction; in fact it’s a true isomerization. The molecular formula for the cyclized form is the same as the open chain form

35. Biochemistry: Vitamins; Carbohydrates I p. 35 of 58 Family tree of aldoses Simplest: D-, L- glyceraldehyde (C3) Add —CHOH: D,L-threose, erythrose (C4) Add —CHOH: D,L- lyxose, xylose, arabinose, ribose (C5) Add —CHOH: D,L-talose, galactose, idose, gulose, mannose, glucose, altrose, allose (C6)

36. Biochemistry: Vitamins; Carbohydrates I p. 36 of 58 Family tree of ketoses Simplest: dihydroxyacetone (C3) Add —CHOH: D,L-erythrulose (C4) Add —CHOH: D,L- ribulose, xylulose (C5) Add —CHOH: D,L-sorbose, tagatose, fructose, psicose

37. Biochemistry: Vitamins; Carbohydrates I p. 37 of 58 Haworth projections …provide a way of keeping track the chiral centers in a cyclic sugar, as the Fisher projections enable for straight-chain sugars Sir Walter Haworth

38. Biochemistry: Vitamins; Carbohydrates I p. 38 of 58 The anomeric carbon In any cyclic sugar (monosaccharide, or single unit of an oligosaccharide, or polysaccharide) there is one carbon that has covalent bonds to two different oxygen atoms We describe this carbon as the anomeric carbon C O O

39. Biochemistry: Vitamins; Carbohydrates I p. 39 of 58  -D- glucopyranose One of 2 possible pyranose forms of D- glucose There are two because the anomeric carbon itself becomes chiral when we cyclize

40. Biochemistry: Vitamins; Carbohydrates I p. 40 of 58  -D- glucopyranose Differs from  - D-gluco- pyranose only at anomeric carbon

41. Biochemistry: Vitamins; Carbohydrates I p. 41 of 58 Count carefully! It’s tempting to think that hexoses are pyranoses and pentoses are furanoses; But that’s not always true The ring always contains an oxygen, so even a pentose can form a pyranose In solution: pyranose, furanose, open- chain forms are all present

42. Biochemistry: Vitamins; Carbohydrates I p. 42 of 58 Substituted monosaccharides Substitutions on the various positions retain some sugar-like character Some substituted monosaccharides are building blocks of polysaccharides Amination, acetylamination, carboxylation common O OH HO HNCOCH 3 OH OHO OH OO-O-

43. Biochemistry: Vitamins; Carbohydrates I p. 43 of 58 Acetals and ketals Hemiacetals and hemiketals are compounds that have an –OH and an –OR group on the same carbon Cyclic monosaccharides are hemiacetals & hemiketals Acetals and ketals have two —OR groups on a single carbon Acetals and ketals are found in glycosidic bonds

44. Biochemistry: Vitamins; Carbohydrates I p. 44 of 58 Sucrose: a glycoside A disaccharide Linkage is between anomeric carbons of contributing monosaccharides, which are glucose and fructose

45. Biochemistry: Vitamins; Carbohydrates I p. 45 of 58 Reducing sugars Sugars that can undergo ring-opening to form the open-chain aldehyde compounds that can be oxidized to carboxylic acids We describe those as reducing sugars because they can reduce metal ions or amino acids in the presence of base Benedict’s test: 2Cu 2+ + RCH=O + 5OH -  Cu 2 O + RCOO - + 3H 2 O Cuprous oxide is red and insoluble

46. Biochemistry: Vitamins; Carbohydrates I p. 46 of 58 Ketoses are reducing sugars In presence of base a ketose can spontaneously rearrange to an aldose via an enediol intermediate, and then the aldose can be oxidized.

47. Biochemistry: Vitamins; Carbohydrates I p. 47 of 58 Sucrose: not a reducing sugar Both anomeric carbons are involved in the glycosidic bond, so they can’t rearrange or open up, so it can’t be oxidized Bottom line: only sugars in which the anomeric carbon is free are reducing sugars

48. Biochemistry: Vitamins; Carbohydrates I p. 48 of 58 Reducing & nonreducing ends Typically, oligo and polysaccharides have a reducing end and a nonreducing end Non-reducing end is the sugar moiety whose anomeric carbon is involved in the glycosidic bond Reducing end is sugar whose anomeric carbon is free to open up and oxidize Enzymatic lengthening and degradation of polysaccharides occurs at nonreducing end or ends

49. Biochemistry: Vitamins; Carbohydrates I p. 49 of 58 Nucleosides Anomeric carbon of ribose (or deoxyribose) is linked to nitrogen of RNA (or DNA) base (A,C,G,T,U) Generally ribose is in furanose form This is an example of an N-glycoside Diagram courtesy of World of Molecules

50. Biochemistry: Vitamins; Carbohydrates I p. 50 of 58 Polysaccharides Homoglycans: all building blocks same Heteroglycans: more than one kind of building block No equivalent of genetic code for carbohydrates, so long ones will be heterogeneous in length and branching, and maybe even in monomer identity

51. Biochemistry: Vitamins; Carbohydrates I p. 51 of 58 Storage polysaccharides Available sources of glucose for energy and carbon Long-chain polymers of glucose Starch (amylose and amylopectin): in plants, it’s stored in 3-100 µm granules Glycogen Branches found in all but amylose

52. Biochemistry: Vitamins; Carbohydrates I p. 52 of 58 Amylose Unbranched,  -1  4 linkages Typically 100-1000 residues Not soluble but can form hydrated micelles and may be helical Amylases hydrolyze  -1  4 linkages Diagram courtesy Langara College

53. Biochemistry: Vitamins; Carbohydrates I p. 53 of 58 Amylopectin Mostly  -1  4 linkages; 4%  -1  6 Each sidechain has 15-25 glucose moieties  -1  6 linkages broken down by debranching enzymes 300-6000 total glucose units per amylopectin molecule One reducing end, many nonreducing ends

54. Biochemistry: Vitamins; Carbohydrates I p. 54 of 58 Glycogen Principal storage form of glucose in human liver; some in muscle Branched (  -1  4 + a few  -1  6) More branches (~10%) Larger than starch: 50000 glucose One reducing end, many nonreducing ends Broken down to G-1-P units Built up from G-6-P  G-1-P  UDP- Glucose units

55. Biochemistry: Vitamins; Carbohydrates I p. 55 of 58 Glycogen structure

56. Biochemistry: Vitamins; Carbohydrates I p. 56 of 58 Structural polysaccharides Insoluble compounds designed to provide strength and rigidity Cellulose: glucose  -1  4 linkages Rigid, flat structure: each glucose is upside down relative to its neighbors 300-15000 glucose units Found in plant cell walls Resistant to most glucosidases Cellulases found in termites, ruminant gut bacteria Chitin: GlcNAc  -1  4 linkages: exoskeletons, cell walls

57. Biochemistry: Vitamins; Carbohydrates I p. 57 of 58 Glycoconjugates Poly or oligosaccharides covalently linked to proteins or peptides Generally heteroglycans Categories: Proteoglycans (protein+glycosaminoglycans) Peptidoglycans (peptide+polysaccharide) Glycoproteins (protein+oligosaccharide)

58. Biochemistry: Vitamins; Carbohydrates I p. 58 of 58 Proteoglycans: Glycosaminoglycans Unbranched heteroglycans of repeating disaccharides One component is GalN, GlcN, GalNAc, or GlcNAc Other component: an alduronic acid —OH or —NH2 often sulfated Found in cartilage, joint fluid