Carbohydrate Structure Notes

Carbohydrate Structure

• Carbohydrates are not as simple as just carbon and water.
• The lecture is presented by Sian Patterson, Ph.D., Associate Professor, Teaching Stream Biochemistry, University of Toronto.
• Examples of sugars include raw sugar and refined sugar.

Learning Objectives

• Explain the importance of carbohydrate structure for biochemical processes.
• Describe the organization of functional groups and how they contribute to carbohydrate complexity (mono vs. polysaccharides).
• Differentiate between the enzymatic synthesis and breakdown of complex carbohydrates.
• State the physiological importance of reducing sugars and explain how they may be detected in the lab.
• Describe how carbohydrates are transported across the plasma membrane and into the cell.

Carbohydrates - Good or Bad?

• Carbohydrates are present in various foods, such as Smarties.
• Smarties ingredients include: Milk chocolate, sugar.

What We Know about Carbohydrates

• Where have you seen sugars in the context of biology and biochemistry?
• Common features of carbohydrate molecules.

Glycosidic Bonds and Sugar Structures

• Glycosidic bonds can form between proteins and carbohydrates.
• Examples of sugar structures:
  • Glucose: monosaccharide
  • Fructose: monosaccharide
  • Sucrose: disaccharide

Brain Break: Sucrose vs. Saccharin

• Sucrose and saccharin both bind to GPCRs, but saccharin is 100X sweeter.
• The question is what accounts for this difference?

Carbohydrate Numbering

• Carbohydrates can be aldoses or ketoses.
• Examples:
  • D-Glyceraldehyde
  • D-Erythrose
  • D-Ribose
  • D-Glucose
  • D-Erythrulose

Carbohydrate Configuration

• L or D is assigned to the asymmetric, chiral carbon furthest away to the carbonyl.
• Two isomers: L or D.
• Chiral carbon furthest away from aldehyde: capital.
• Two possible linear stereoisomers.

Carbohydrate Cyclization

• Aldehydes, ketones, and hydroxyl groups are very reactive functional groups.
• The aldehyde/ketone carbonyl undergo nucleophilic attack by hydroxyl groups.
• Intramolecular reactions for a 5-member furan ring or a 6-member pyran ring.
• The new bond may be a:
  • Hemiacetal: aldehyde derivative
  • Hemiketal: ketone derivative

D-Glucose

• D-Glucose can exist in open-chain and cyclic forms.
• The cyclic forms include α-D-Glucopyranose and β-D-Glucopyranose.

Anomeric Carbons are Chiral

• Haworth projections let you ‘see’ cyclic sugars in 3D.
• Intramolecular cyclization is reversible in solution through the linear chain.
• The chirality of cyclic structures can be detected by mutarotation; a change in the optical rotation of polarized light, following the interconversion between the α and β anomer.
• The reactivity of aldehydes (and ketones) also means sugars can react with other molecules (intermolecular reaction) but only in the linear form.

Glycation example - HbA1c

• Glucose is highly reactive in the linear form.
• Glucose in the blood will react with the N-terminal Valine on hemoglobin, producing hemoglobin A1c (HbA1c), a marker of high blood glucose.
• Ranges (Diabetes Canada):
  • Normal: < 6%
  • Prediabetes: 6-6.4%
  • Diabetes > 6.4%
• Other molecules (pr/lipid/DNA) in the body can also be glycated, which may affect their function.
• Initial glycation reaction. The intermediate products are known as Schiff base, Amadori, and Maillard products.
• Glycation involves covalent reactions between free amino groups of amino acids, such as lysine, arginine, or protein terminal amino acids and sugars (glucose, fructose, ribose, etc), to create, first, the Schiff base and then Amadori products, of which the best known are HbA1c and fructosamine (fructoselysine).
• AGE formation from fructoselysine involves the nonoxidative dissociation of fructoselysine to form new reactive intermediates that again modify proteins to form AGEs of various different chemical structures.
• Alternatively, fructoselysine decays and releases its carbohydrate moiety either as glucose or as the more reactive hexoses, such as 3-deoxyglucosone, which themselves may modify proteins. In addition, it has recently been found that glucose can auto-oxidize to form reactive carbonyl compounds (glyoxal and methylglyoxal) which can react with proteins to form glycoxidation products.
• In addition to this, products of oxidative stress, such as peroxynitrite, can also induce the formation of carboxymethyl lysine by oxidative cleavage of Amadori products and/or the generation of reactive dicarbonyl compounds from glucose.
• Accumulation of AGEs in the ECM occurs on proteins with a slow turnover rate, with the formation of cross-links that can trap other local macromolecules. In this way, AGEs alter the properties of the large matrix proteins collagen, vitronectin, and laminin. AGE cross-linking on type I collagen and elastin causes an increase in the area of ECM, resulting in increased stiffness of the vasculature. Glycation results in increased synthesis of type III collagen, type V collagen, type VI collagen, laminin, and fibronectin in the ECM, most likely via upregulation of transforming growth factor-β pathways. Formation of AGEs on laminin results in reduced binding to type IV collagen

Honey - Isn’t it sweet?

• The main sugar found in honey is fructose.
• Also contains glucose, sucrose, water, and trace amounts of other molecules.
• Fructose can form a furan ring as well as a pyran ring.
• Heating sugars can affect their structure and properties, i.e., sweetness.

Reducing Sugars

• Cyclic sugars need a free OH to convert back to a C=O to yield a positive reducing test.

Brain Break: Reducing Sugars?

• Examples of molecules with OH and CH_2OH groups

Why are the different positions important?

• Glucokinase is an example.
• The active site of Glucokinase is shown.
• K_m Glucose = ~6-10 mM
• K_m Galactose = Not Measurable

Simple Monosaccharides

• Monosaccharides are the simplest carbohydrates (C ildewide H2O)n.
• Carbohydrates are aldoses (C1-aldehyde) or ketoses (C2-ketone).
• Chiral carbons (x) lead to structural diversity where 2^x determines the number of linear stereoisomers.
• D - or L - is decided by orientation around asymmetric carbon furthest from aldehyde/ketone. D-sugars are biologically important!
• Cyclization results in the formation of 2 additional structures.

Carbohydrate Terminology

• Isomers: same formula
  • Constitutional isomers: diff struct. different order of functional group.
  • Stereoisomers: same formula and order
    • Enantiomers: non superimposable mirror images
    • Diastereoisomers: non mirror images
      • Epimers: Differ at one asymmetric carbon
      • Anomers: Rings that differ at new assymetric carbon

Carbohydrate Modification

• Sugars can be phosphorylated, methylated, or N-containing functional groups may be added.
• Hydroxyls (or even the carbonyl) may be removed.
• This increases the complexity of carbohydrate structure.

Complex Carbohydrates

• Monosaccharides – 1 sugar
• Disaccharides – 2 sugars
• Oligosaccharides – 3 to 20
• Polysaccharides – up to 1000s

Glycosidic bonds

• Monosaccharides are joined by glycosidic linkages to form disaccharides and more complex structures.
• Glycosidic bond formation is a condensation reaction where \text{H}2 ildewide ildewideO is lost. Cleaving glycosidic bonds requires \text{H}2 ildewide ildewideO.
• Intermolecular glycosidic bonds are formed between a hydroxyl or amine and a reactive anomeric carbon on another molecule.
• Di- = 2, Oligo- = 3-20+, Poly- = up to 1000s of units
• Glycoproteins may be O-linked (Ser/Thr) or N-linked (Asn).
• Hydrolase

Disaccharide bond formation

• Diagram of disaccharide bond formation.

Variation on the O-glycosidic bond

• Formation of Methyl α-D-glucopyranoside + \text{H}_2 ildewide ildewideO

Variation #2 - Glycosylation in Proteins

• Glycoproteins may be O-linked (Ser/Thr) or N-linked (Asn).
• GlcNAc
• β-Galactosyl-(1-3)-α-N-acetylgalactosaminyl-Ser/Thr

Sugar sequencing isn’t easy due to the complex structures that can form.

• What are 4 things that can vary in an oligosaccharide?

Sugar Sequencing

• Sugar sequencing is much more complicated due to the presence of different isomers.
• Polysaccharides differ in:
  • Composition: Glc vs. Gal
  • Connectivity: 1→4, 1→6
  • Configuration: α vs. β

Cleavage to Monosaccharides

• Cells can only transport and use monosaccharides for fuel.
• Enzymes can breakdown and release monosaccharides from di/oligo/polysaccharides.
• Lactase, maltase, and sucrase are enzymes that cleave specific disaccharides using \text{H}_2 ildewide ildewideO.
• These enzymes are found in your saliva and the microvilli of the small intestine to help with monosaccharide absorption.

Protein Recognition of Sugars

• Amino acid side chains in the active sites of enzymes confer specificity through non-covalent interactions.

Disaccharides

• Maltase cleaves the Maltose to Glucose + Glucose
• Lactase cleaves the Lactose to Galactose + Glucose

Polysaccharides

• Large sugar polymers – 1000s of monosaccharides, linked in a linear or branched fashion.
• Important for energy storage, cellular structure, and recognition.
• Also known as glycans.
• Homopolymer: same monosaccharides. – e.g., glycogen and starch (homoglycans).
• Heteropolymer: different monosaccharides. – e.g., sugars found in glycoproteins (heteroglycans).

Glycosidic Linkages found in Starches

• Detectable using Iodine.
• Amylose:
  • unbranched glucose units
  • α(1→4)-linkages
• Amylopectin:
  • linear glucose chains joined by α (1→4)- linkages.
  • α (1→6)-linkages at branch points once every 30 glucose units.

Starch and Iodine

• The image shows starch interacting with iodine.

Enzymatic Digestion of Starches

• α-amylase is secreted by the salivary glands and pancreas and cleaves at random locations along the chains to give maltose and maltotriose.

Cellulose

• The most abundant organic compound, serving a structural role in plants.
• Unbranched chains of glucose units are joined by β(1→4) linkages with many hydrogen bonds.
• Cellobiose is a disaccharide of glucose linked by β1→4.
• Negative iodine test due to a different macromolecular structure.
• Humans don’t produce cellulase.

Complex Polysaccharides - Glycogen

• A storage form of long, branched chains of glucose.
• Contains branch points every 8-12 glucose units.
• Contains a dimer of glycogenin at the centre.
• Glucose units are added and removed from the non-reducing ends by enzymes.
• Found in the liver and muscle.

Glycogen

• Diagram of Glycogen structure showing α-1,4-glycosidic bonds and α-1,6-Glycosidic bonds.

Starch and Glycogen

• Diagram showing Starch and Glycogen

Glucose Transport

• Moving glucose around the body is critical.
• Glucose is a very polar molecule and cannot diffuse through the lipid bilayer.
• Insulin helps signal for the recruitment of some sugar transporters (GLUT4) to the plasma membrane.
• Different cells will use different glucose transporters and different mechanisms:
  • Sodium-Glucose co-transporters (SGLTs) - secondary active transport
  • Glucose Transporters - facilitated diffusion
  • Fructose Transporters - facilitated diffusion

Glut Transporters

• Question about the binding affinity of different glucose transporters.

Key Messages

• Carbohydrates take on a variety of complex structures.
• Sugar structure is crucial for enzymatic recognition and plays a role in many biochemical and cellular processes.
• Monosaccharides can be linear chains or form cyclic structures, and their reactivity also allows them to combine to form higher order structures or react with other molecules.
• Polysaccharides are carbohydrate storage molecules to be used for fuel or for structural purposes.
• Enzymes assist in import and formation or breakdown of oligosaccharides.