CARBOHYDRATES. lectures 11, 12 & 13. Docent Tuomo Glumoff. Faculty of Biochemistry and Molecular Medicine P Biomolecules for Biochemists (8 op)

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1 Faculty of Biochemistry and Molecular Medicine P Biomolecules for Biochemists (8 op) P Biomolecules for Bioscientists (8 op) P Biomolecules (5 op) lectures 11, 12 & 13 CARBOYDRATES Docent Tuomo Glumoff

2 CARBOYDRATES lectures 1 Figures under taken from the following sources: Devlin: Textbook of Biochemistry with Clinical Correlations orton et al., Principles of Biochemistry Nelson & Cox: Lehninger Principles of Biochemistry Mathews et al., Biochemistry Voet & Voet: Biochemistry Berg et al., Biochemistry Campbell et al., Biochemistry Illustrated Varki et al. (ed.) Essentials of Glycobiology Cooper & ausman: The Cell A Molecular Approach Rawn: Biochemistry Brown: Biochemistry Appling et al., Biochemistry concepts and connections Answers to problems will be given at the lectures and they will also be available after the lectures in NOPPA. 1. Basics of carbohydrates - carbohydrates = saccharides sugars - What is a sugar? ow to define a sugar? Typical properties of sugars? sweet taste crystallizes water-soluble polyhydroxy aldehydes or polyhydroxy ketones contains a chiral carbon optically active (stereoisomers) for monosaccharides the molecular formula is (C 2 O) n, where 3 n 9 O O O O O O O O O O O O O O O *) (C 2 O) n is an oversimplification, since many saccharides are modified and some contain also atoms such as S and N. Nevertheless, all compounds herewith either have this formula or can be derived from substances that do. (Mathews et al. Biochemistry 4th edition) PROBLEM 1. Why a 2-carbon compound cannot be a sugar although it would fulfill the formula (C 2 O) n? - most abundant class of biological molecules on earth by mass - all organisms can synthesize, but most important is photosynthesis: solar energy to chemical energy - mono- and disaccharides are usually metabolites - oligosaccharides are usually linked to proteins or lipids - polysaccharides are usually storage forms or have structural function

3 2. Monosaccharides 2 Monosaccharides - trioses, 3 carbons - ketoses and aldoses are tautomers (an aldotriose) (a ketotriose) PROBLEM 2. Please check that the formula (C 2 O) n hold for both triose tautomers! - D- and L-enantiomers = isomers differing at the chiral carbon configuration mirror images - in nature D-monosaccharides dominate

4 - D-L vs. R-S naming system: R- and S- would be absolute, but becomes difficult with multiple chiral carbons 3 +/- and d/l (to right vs. to left) could be used with respect to ability to rotate planepolarized light PROBLEM 3. You will be given molecular models of an R-isomer and an S-isomer. Which is which? red = blue =

5 - tetroses, 4 carbons - may have 2 chiral carbons, and thus are diastereomers - D- or L- is assigned based on the chiral carbon farthest from the carbonyl group (= highest numbered chiral C) and additional names are given for diastereomers (an arbitrary naming system) - epimers = multiple D/L possibilities, but a difference in only one of them 4 - pentoses, 5 carbons for example riboses in nucleotides - hexoses, 6 carbons typical energy molecules, like glucose D-sugars have the same absolute configuration at the asymmetric center furthest from their carbonyl group as does D-glyceraldehyde Examples of monosaccharides with their occurrence and physiological role: PROBLEM 4. Observe the aldose and ketose structures on page 5 and answer the following questions: 4.1 Why are there a fewer number of different ketoses? 4.2 Identify each of the following: a) two aldoses whose configuration at carbons 3, 4 and 5 matches that of D-fructose b) the enantiomer of D-galactose c) an epimer of D-galactose that is also an epimer of D-mannose d) a ketose that has no chiral centers e) a ketose that has one chiral center

6 Fischer projections of C3 to C6 aldoses 5 carbon numbering: Fischer projections of C3 to C6 ketoses

7 - the open chain form of a monosaccharide is in equilibrium with the ring form - under physiological conditions 5-carbon monosaccharides are 99% in the ring form 6 Pyranose formation: The open-chain form of glucose cyclizes when the C-5 hydroxyl group attacks the oxygen atom of the C-1 aldehyde group to form an intramolecular hemiacetal. Two anomers, designated a and b, can result. - aworth projection: thick edge towards the viewer, thin edge towards the rear - interconversion between a and b forms = mutarotation - catalyzed by specific mutarotases trans cis a = on the opposite side b = on the same side

8 hemiacetal or hemiketal bond is formed - pentoses and hexoses form pyran and furan rings => pyranoses and furanoses - anomers = upon cyclization the former carbonyl carbon becomes asymmetric => anomeric carbon

9 - note that pentoses and hexoses may both form 5- and 6-rings (size of ring not dependent on number of carbons!) - distribution between furanose and pyranose depends on the sugar, p, solvent and temperature 8 * * glucose: open chain form 0.2 %

10 PROBLEM 5. On page 10 you will find the aworth projection of b-d-nacetylglucosamine. Draw it using the Fischer projection (open-chain form). PROBLEM 6. Take mannose from the tables on page 5 and draw it in a ring form. 9 chair and boat conformations: - same stereochemistry, difference in three-dimensional shape - conformations with bulky substrates in equatorial positions are favored, while steric hindrance of axial substituents renders the boat conformation less favored e = equatorial a = axial - conformations: interchangeable by simple deformation of the molecule - configurations: interchangeable only by breaking and reformation of covalent bonds

11 - monosaccharides may contain substituents or modifications that make them derivatives - examples: 10 - aminosugars: - reduction of the carbonyl group => alditol (xylitol is accordingly made from xylose) glyceraldehyde-3-phosphate glucose-1-phosphate - sugar phosphates are very important derivatives - activated molecules in energy metabolism glucose-6-phosphate fructose-6-phosphate

12 - glycosides: a glycosidic bond is formed 11 - glycosidic bond is the bond between two sugar units in di- and polysaccharides, but as seen here the glycosidic bond can also form between a sugar and another group - glycosidic bond is also present in nucleotides in DNA and RNA - common abbreviations used for monosaccharides

13 12

14 3. Disaccharides 13 - two monosaccharides are joined with a glycosidic bond => hydroxyl group of one sugar reacts with the hydroxyl group of the anomeric carbon of another sugar NOTE!! - equilibrium to left - polysaccharides are metastable compounds - hemiacetal becomes acetal - in common disaccharides O-glycosidic bond; in glycoproteins and nucleotides N-glycosidic bond - glycosidic bond is readily hydrolyzed in acid, but resists bases - oxidation of the anomeric carbon means that the sugar is a reducing sugar (remember: oxidation and reduction are always coupled the compound that gets oxidized will leave another compound reduced) - a reducing sugar has a reducing end and a nonreducing end - reducing end is the one with a free anomeric carbon (and thus also a free aldehyde group) - only the open-chain form can undergo oxidation, not the hemiacetal (ring) form - in a disaccharide only the rightmost monomer can adopt the open chain form

15 14 - mutarotation may also take place in disaccharides, namely the reducing end monosaccharide - a,a form is simply a, while a,b is simply b - if the disaccharide ends in a nonreducing end, it cannot mutarotate (such as sucrose) Practical example of using Copper reduction by a reducing sugar in clinical biochemistry: - amount of glucose in blood or urine (Fehling s test) - e.g. insulin dosage control - but modern tests are based on specific enzymes acting on glucose! PROBLEM 7. One of the disaccharides on the following page is not a reducing sugar? Which one and why?

16 Distinguishing features of different disaccharides: The two monomers may be of the same kind, or they may be different 2. The most common linkages between two monosaccharides are: (at least one anomeric hydroxyl is always involved in the bond) 3. The order of the two monomers (if different) determines if the disaccharide can be a reducing sugar or not. 4. The anomeric configuration of the hydroxyl group on carbon 1 of each monomer determine which enzyme can hydrolyze the glycosidic bond (maltose and cellobiose are both made up from two glucoses, but cannot serve as a substrate for the same hydrolytic enzyme) - Why? Disaccharides in 3D for example from here: Compare maltose with cellobiose! Nomenclature of disaccharides (systematic names): Reducing disaccharides (contain a free hemiacetal): "glycosylglycose" example: α-d-glucopyranosyl-(1 4)-β-D-glucopyranose (trivial name β-maltose) Non-reducing disaccharides (without a free hemiacetal): "glycosyl glycoside" example: α-d-glucopyranosyl- (1 1)- α-d-glucopyranoside (trivial name α,α-trehalose)

17 Names and writing of disaccharides: - sequence starts from the left (nonreducing end) - anomeric and enantiomeric forms can be designated - the ring configuration can be given - the atoms between which the glycosidic bond is formed are given (the arrow shows the direction from the anomeric carbon) maltose: sucrose: a-d-glcp(1 4)-b-D-Glcp or Glc(a1 b4)glc a-d-glcp(1 2)-b-D-Fruf "invert sugar" = hydrolysis of sucrose to glucose and fructose 16 PROBLEM 8. Write below a systematic name for lactose. 4. Polysaccharides = glycans - storage polysaccharides like, starch and glycogen, as well as stability, biosynthesis and degradation of polysaccharides will be postponed to Aineenvaihdunta I (Metabolism I) course later in the spring - here we will study other types of oligo- and polysaccharides and their functions - homopolysaccharides repeating one type of units - heteropolysaccharides repeating usually two kinds of units - complex polysaccharides contain more than two kinds of units

18 - structural elements in plant cell walls and animal exoskeletons - extracellular support bacterial cell envelope and in animals a matrix that holds cells together and supports tissues and organs - no defined molecular weight = saccharide chains may grow to become shorter or longer (But NOTE: saccharide chains with specific function e.g. in recognition must be of certain length and composition; see later blood group antigens) 17

19 Cellulose: - linear polymer of b-d-glucose, to units - most abundant polysaccharide molecule on earth 18 But NOTE: saccharide chains with specific function e.g. in recognition must be of certain length and composition; e.g. blood group antigens specific difference Chitin: - linear polymer of b-n-acetylglucosamine - found in surface armor of insects Glycosaminoglycans: - polymers of repeating disaccharide units - one of the sugars is either N-acetylgalactosamine or N-acetylglucosamine or a derivative thereof - are acids through either a sulfate or carboxylate group present - examples of modified sugar residues that gain novel properties and functions - heparin is a natural anticoagulant in body fluids (here only the repeating unit shown!) - inhibits blood clotting enzymes through binding to anti-prothrombin III protein PROBLEM 9. Can you think of a practical application for heparin in clinical biochemistry?

20 19 GlcUA GlcUA

21 - hyaluronic acid is noted for its much longer chain than most other glycosaminoglycans 20 PROBLEM 10. Chain length of a polysaccharide (number of units in a chain) is not known in the beginning of its synthesis, i.e. it is not predefined. See p. 19: the numbers are not precise sometimes the glycan chain is shorter, sometimes longer. Please explain!

22 21 Cooper & ausman: The Cell crosslinked tetrapeptides Voet et al.: Principles of Biochemistry

23 Glycoconjugates: proteoglycans, glycoproteins and glycolipids - proteoglycan is a protein-carbohydrate complex found in extracellular space - peptidoglycan is an important part of bacterial cell walls 22 Left: Gram negative bacilli Right: Gram positive cocci

24 - for comparison, the cell wall of mycobacteria is even more complicated in structure (figure below) - on top of the peptidoglycan layer there are arabinogalactan (carbohydrate) and mycolic acid (lipid) layers - this makes the cell wall more resistant and contributes to the difficulty of eliminating tuberculosis causing mycobacteria 23

25 24

26 25 galectin = galactoside-binding lectin lectin = sugar-binding protein - an example, how membrane proteins and carbohydrate structures attached to them form part of the cell s contact network - cell to cell - cell to extracellular matrix - protein-protein interactions - protein-carbohydrate interactions - cell surface structures mediated via carbohydrates can be part of intracellular signaling

27 - glycoproteins are proteins that contain certain types of carbohydrate chains attached to them - O-linked glycoproteins and N-linked glycoproteins 26 - N-linked glycans are attached to asparagine residues in a sequence Asn X Ser/Thr - O-linked glycans are attached to threonines or serines - glycan chains can be of various composition and type of branching - glycans are attached to protein cotranslationally in the ER (endoplasmic reticulum) - glycans are further modified in the Golgi apparatus - glycan chains give the cellular machinery extra possibilities for recognizing proteins and cells immunoglobulin tissue distribution and interaction with phagocytic cells intracellular targeting and excretion cellular identification control of body fluid viscosity blood groups - other functions of glycans include: stabilize protein fold (ready-made protein) stabilize protein folding (during the folding!) by binding to intermediate conformations stiffens and extends the polypeptide chain

28 - note that saccharide chains are actually surprisingly large in size compared to proteins a chain of a couple of sugar residues easily make a structure with some dimensions comparable to a protein molecule PROBLEM 11. E. coli is often used in the lab to produce cloned eukaryotic proteins. Sometimes the proteins cannot be obtained in good amounts (there can be many reasons). Can you identify or guess one possible reason from the material we just discussed? 27 - oligosaccharide chains attached to membrane proteins at the surface of red blood cells - to attach the terminal monosaccharide to get either A or B blood group, one needs specific enzymes; heterozygotes have both to get AB - type O oligosaccharides are nonantigenic PLEASE NOTE!!!! (refer to page 18) Whenever a glycan chain has a specific recognition function, the number and the sugar structure must be precise!!!!!

29 W.W. Christie The Scottish Crop Research Institute, Dundee - glycolipids and lipopolysaccharides are membrane components, where carbohydrates are attached to lipids 28 - lipopolysaccharides (LPS) can be found on the surface of bacteria, like E. coli and Salmonella - antibodies are raised in the body against LPS to fight bacterial infections - LPS of some bacteria are toxic Lipid layer PROBLEM 12. Write down the distinctive differences of the cell wall structures between Gram-positive and negative bacteria. Good to be aware of for possible further need: Nomenclature of Carbohydrates: INTERNATIONAL UNION OF PURE AND APPLIED CEMISTRY (IUPAC) and INTERNATIONAL UNION OF BIOCEMISTRY AND MOLECULAR BIOLOGY (IUBMB) Joint Commission on Biochemical Nomenclature (JCBN) Symbols for Specifying the Conformation of Polysaccharide Chains:

30 5. Briefly about importance of carbohydrate-acting enzymes 29 - glycan binding proteins are abundant due to large amount of different carbohydrate structures - for the synthesis or degradation of various glycan molecules a large amount of specific enzymes are needed - specific for a type of bond and sugar molecules connected by the bond - wrong type of carbohydrate structures are oftentimes found in cancer cells

31 6. Some extraordinary di- and oligosaccharides 30 (taken here just as examples of some practically important and interesting applications, but not for exam): - the hydroxy group in a sugar ring may also be derivatized by another sugar ring, like in melezitose, which is a sugar found as a minor component in honey - sugar rings may also form ring-like structures = cyclodextrins - a-, b- and g-cyclodextrins have 6, 7 or 8 glucose units, respectively - cyclodextrins find use in applications like protecting aroma molecules in foodstuffs or transferring and delivering (slowly) drugs in the body due to complex formation with such smaller molecules - streptomycin, an antibiotic, interferes with bacterial protein synthesis

32 - bacterially produced dextrans can be cross-linked to form very hydrophilic preparations that swell and form gels - for example Sephadex, which is used as a chromatographic support media in gel filtration (protein purifications) 31 - Sucralose is an artificial sweetener Stevioside (steviol glycoside) - natural sweetener extracted from leaves of stevia plant times sweeter than glucose - both react with the taste receptors of the tongue like sugars do, i.e. is sweet - zero calories, because cannot be metabolized in the body