Carbohydrates: Glycolysis, Glycogenolysis and important carbohydrates

Carbohydrates are an important class of biomolecules comprising of aldehydes or ketones with multiple hydroxyl groups that is, they are poly hydroxy aldehydes or ketone. Check out my blog post which depicts a brief understanding of carbohydrates.

Function of carbohydrates

• They store energy and act as fuels and important metabolic intermediates. Carbohydrates are important sources of energy, for instance, individual glucose molecules make up the starch found in rice, which breaks down to be eventually used in the generation of ATP
• Carbohydrates like ribose and deoxyribose form the structural framework of nucleic acids like DNA and RNA
• Carbohydrate polymers known as glycans are structural elements in the cell wall of plants and bacteria eg. cellulose, the most abundant organic compound in nature
• Complex carbohydrate polymers like glycoconjugates play key roles in mediating cell to cell interactions through their linkage with proteins and lipids
  

Properties of carbohydrates

Monosaccharides

• Simplest form of carbohydrates are monosaccharides: 
  • Most carbohydrates have an empirical formula of (C-H20)n.  Some may even contain nitrogen, phosphorous or sulphur
  • Monosaccharides have a single aldehyde or ketone group. However, they may have two or more hydroxyl groups. The most abundant monosaccharide in nature is glucose, also known as dextrose
  • They are colourless crystalline solids soluble in water and insoluble in non polar solvents
  • In the open chain form, one of the carbon is double bonded to oxygen forming a carbonyl group and the other carbon atoms attaches to hydroxyl group. If a carbonyl group is present at the end of the carbon chain, it is an aldehyde. If it is in the carbon chain, it is a ketone
  • Smallest and simplest monosaccharides called trioses have n=3 are dihydroxyacetone (with a ketone group) and glyceraldehyde (with an aldehyde group)
  • Modifications of monosaccharides: upon reactions with alcohols and amines, they can form adducts. Eg:

Enantiomers

• Glyceraldehyde, due to its chiral carbon exists in 2 enantiomers: l and d. In fact, many monosaccharides have chiral centres giving rise to many sugar sterioisomers in nature. One monosaccharide which does not have a chiral carbon is dihydroxyacetone
• Tetroses, pentoses, hexoses and heptoses are monomers where n=4,5,6 and 7 respectively. They have many asymmetric or chiral carbon, so they exist as diastereoisomers (not mirror images of each other)

Epimers

• Epimers are sugars differing in configuration at a single asymmetric carbon eg. d- glucose and d- mannose differ at C2, d- galactose and d- glucose differ at C4

Cyclic forms

• Most sugars in the solution are not in open chains, especially sugars having more than four carbons. They rather cyclise into closed structures or rings. Aldehyde reacts with alcohol to form hemiacetal and ketone reacts with alcohol to form hemiketal
• Sugars with aldehydes form a pyranose ring ( 6 membered ) by the reaction of C1 of aldehyde and C5 of hydroxyl group
• Sugars with ketones form a furanose ring (5 membered) by the reaction of C2 of keto group and C6 of hydroxyl group
• Anomers: the formation of a cyclic hemiacetal generates an additional asymmetric center. For instance, in glucose, C1 forms the asymmetric centre giving rise to alpha-d-glucopyranose and beta-d-glucopyranose. In this case, anomeric carbon is C1 and anomers are the 2 forms. The alpha and beta glucopyranose interconevert in an aqueous solution through a process called mutarotation
• Chair and boat form: the pyranose and furanose rings are not planar due to its tetrahedral geometry. So, they take 2 forms- chair and boat. The propensity towards chair and boat form depend on the stearic hindrance
• Furanose rings take up a puckered conformation called envelope

Reducing agents

• Monosaccharides as reducing agents: due to the presence of carbonyl group, it gets readily oxidized to a carboxyl group. Glucose and other sugars which can reduce cupric ion are reducing sugars. They form enediols which get converted aldonic acid and a complex mixture of acids. The biochemical test to identify reducing sugars is feelings test. However, note that this reaction is possible only in its open chain form and not in its cyclic form as it lacks its reducing end (the end with a free anomeric carbon)

anomeric carbon of glucose + hydroxyl group of methanol= alpha d glucopyranoside and beta d glucopyranoside (both anomers)

• Glycosidic bond forms between the anomeric carbon and hydroxyl group, specifically O glycosidic bond. If anomeric carbon is linked to nitrogen of an amine group, it is called N glycosidic bond

• Another classic example is adducts formed between sugars (like ribose or deoxyribose) and amines (like adenine) to give nucleosides

Complex carbohydrates

• The presence of many hydroxyl group can help link monosaccharides together through glycosidic linkage. They can form disaccharides, oligosaccharides or polysaccharides
• For instance, maltose is a disaccharide made of 2 molecules of glucose through alpha 1,4- glycosidic bond ( C1 of one sugar and hydroxyl oxygen atom of C4)
• Complex carbohydrates are readily hydrolysed by acids while resisting cleavage from bases. So, boiling them in dilute acids can help hydrolyse a disaccharide or oligosaccharide

Disaccharides

• Made of 2 monosaccharides bound through O- glycosidic linkage, they are the most abundant oligosaccharides in nature
• Common disaccharides- sucrose, lactose, maltose
• While lactose and maltose are reducing sugars, sucrose is a non reducing sugar as it’s anomeric carbons are involved in glycosidic bond formation and therefore does not have a reducing end
  • Glucose + Fructose= Sucrose , commercially synthesised from beets or canes. Enzyme Sucrase can cleave sucrose to glucose and fructose
  • Glucose + Galactose= Lactose, beta 1,4-glycosidic linkage, disaccharide of milk, hydrolysed by lactase in humans and beta galactosidase in bacteria
  • Glucose + Glucose= maltose, alpha 1,4 glycosidic linkage, cleaved by maltase
• The outer surface of epithelial cells in the small intestine contains these three enzymes

Polysaccharides 

• Also known as glycans, they form through the linkage of multiple monosaccharides
• They are usually formed by 20 or more monosaccharide units. They maybe linear (like cellulose) or branched (like glycogen)
• They play crucial role in energy storage and maintaining the structural integrity of an organism
• They do not have a defined molecular weight, unlike proteins. This is because of the mechanism of assembly of two types of polymers

Types of polysaccharides: 

• Homopolysaccharides: if all monosaccharides are the same
• Heteropolysaccharides: 2 or more types of monosaccharides make up heteropolysaccharides
• Most common homopolymer in animals- glycogen, which stores glucose. The alpha 1,4-glycosidic bonds link the glucose units and alpha 1,6-glycosidic links the branches formed once every 10 units
• Counterpart of glycogen (nutritional reservoir) in plants- starch. It exists in 2 forms: unbranched amylose (alpha 1,4 linkage) and branched amylopectin (alpha 1,6 linkage). Both amylose and amylopectin are cleaved by amylase, secreted by salivary glands and pancreas
  

Important peptides

Glycosaminoglycans

• Collagen, along with other proteins are a main component present in the extracellular spaces of connective tissues such as cartilages, tendons, blood vessels etc. They form a gel like matrix and largely composed of glycosaminoglycans. They are slimy and mucous-like giving them their characteristic viscous and elastic nature
• They are unbranched and consist of alternating residues of uronic acid and hexosamine

Hyaluronate

• Hyaluronate, a glycosaminoglycan acts as a shock absorber and lubricant. They are important components of synovial fluid, vitreous humour and connective tissues, consisting of about 80 to 8000 disaccharide units made of alternating units of glucoronic acid and N acetyl-D-glucosamine (GlcNAc) joined by beta 1,3 bond
• A counter part of hyaluronate in plants is pectin which acts as a shock absorber consisting of galacturonate residues interspersed with rhamnose
• The viscosity of hyaluronate solutions depend on the shear stress. They are highly viscous at low shear rate and the viscosity decreases as the sheer rate increases
• An important property of hyaluronate is that it is secreted by the naked mole rat cells. They secrete extremely high molecular mass and contain up 32000 disaccharide units. This interferes with the signalling pathways that promote the formation of a malignancy. 

Chondroitin sulfate

• Some other common glucosamines like  chondroitin sulphates are sulphated. They differ from the other group of glucosamines by their sulfation of N acetylgalactoseamine residue (GalNAc). Derivatives of chondroitin include derma tan sulphate, keratin sulphate and heparin
• In addition to their presence in connective tissues, joints and synovial fluids, glycosaminoglycans form a part of biofilms. Outside the laboratory, bacteria grows on a surface as a biofilm i.e. an association of cells in a semisolid matrix the matrix consists of highly hydrated polysaccharides like anionic poly-D-glucoronate, poly-N-acetylglucosamine, cellulose and acetylated glycans. The formation of this layer prevents the bacterial cell from washing away

Proteoglycans 

Structure

• They are glycosaminoglycan containing proteins. Both proteins and glycosaminoglycans aggregate through covent and non covalent bonds to form proteoglycans. The structure of a proteoglycan resembles a bottle brush with fine bristles non covalently attached to a hyaluronate backbone. The bristles contain protein in its core surrounded by glycosaminoglycans like keratin and chondroitin sulphate are covalently attached. A link protein attaches the core protein with the hyaluronate
• The entire structure of proteoglycans form highly hydrated gels. This is generally seen in cartilages consisting of collagen fibrils filled with proteoglycans. Upon application of pressure, the cartilage squeezes water away from the proteoglycans until the charge-charge repulsion prevent further compression. Upon the release of pressure, water returns. In fact, the flow of liquids caused by body movements is what nourishes the cartilage in the joints as they lack a blood vessel. This is why upon long periods of inactivity, the cartilage becomes thin and fragile

Location

• They are seen on the cell surface or extracellular matrix where one or more sulphated glycosaminoglycan chains attach either to a membrane protein or secreted protein. They can also bind to extracellular proteins through electrostatic interactions
• The point of attachment of the glycosaminoglycan to the core protein is a serine residue to which the glycosaminoglycan joins through a tetrasaccharide bridge. The serine residue is generally in the sequence Ser-Gly-X-Gly
• In addition to binding core proteins, glycosaminoglycans can also interact with a variety of ligands and can thereby bring about  the interaction of ligands and cell surface receptors. 
• Some proteoglycans can also form aggregates which contribute to the high tensile strength and resilience e of the connective tissue

Sialic acid:

• In addition to proteins , lipids too can have complex oligosaccharide chains. Ganglioside, for examples, is a membrane lipid in which the polar head group is a complex oligosaccharide containing sialic acid in addition to other monosaccharides. 
• It is also called N-acetylneuraminic acids (Neu5Ac) or NANA
• They are a crucial component or glycoproteins and glycolipids

Physiological function

• Due to their negative charge and hydrophilicity, they contribute to important physiological functions in the body. For instance, they are a component of the erythrocytes and contribute to the determination of blood group
• They also help protect oligosaccharides from uptake and destruction by hepatocytes. Proteins called lectins recognize oligosaccharides for uptake and destruction

Lectins

• Lectins have a moderate to high affinity to carbohydrates and bind to them
• Their wide range of functions include: cell-cell recognition, signalling and adhesion. They are also involved in intracellular targeting of newly synthesised proteins
• In plants, they serve as deterrents from insects

Physiological function

• Lectins recognise Leutinizing hormone and thyrotropin in the hepatocytes and help in the uptake of these hormones for destruction, thereby reducing their concentration in the blood
• Certain types of lectins play an important role in protein sorting. For instance, a newly synthesised protein in the ER already has a complex oligosaccharide attached, which can be bound by either of the two lexins. They may either be calnexin (membrane bound )or calreticulin (soluble)

Gluconeogenesis

Why gluconeogenesis?

• Having read a lot about carbohydrates: their structure and properties, one of the most important carbohydrate is glucose, the universal fuel and building block of energy from bacteria to humans
• Fun fact: the brain alone requires 120 grams of glucose per day! And more than half of this is stored as glycogen
• Despite the storage of glycogen as a source of glucose, it is not sufficient under certain conditions. For instance, glycogen depletes quickly between meals or longer fasts or vigorous exercises
• In these cases, our body has to synthesise glucose from non carbohydrate sources through a process called gluconeogenesis

Where does it take place?

• Gluconeogenesis for the most part takes place in the liver. It also takes place in the renal cortex in kidney sometimes
• Cori cycle: during a vigorous exercise, anaerobic glycolysis produces lactate in the skeletal muscle, which is taken up by the liver to convert into glucose. The glucose moves back into the muscle and eventually converts to glycogen. This circuit is called cori cycle
• The non carbohydrate sources for gluconeogenesis include glycolysis products like pyruvate and lactate, citric acid cycle intermediates and carbon skeletons of most amino acids. But, all of these must first convert to oxaloacetate, a four carbon compound
• Basically, gluconeogensis is a pathway by which pyruvate converts to glucose. Most of the reactions of gluconeogenesis are reactions of glycolysis in reverse.  Before heading into gluconeogenesis, let’s have a look at glycolysis. 

Glycolysis pathway in brief

Phase	Substrate	Enzyme	What does the enzyme do	Enzyme requires	Product	Reversible or irreversible
Preparatory phase	Glucose	Hexokinase	Phosphorylates	ATP	Glucose-6-phosphate	Irreversible
	Glucose-6-phosphate	Phosphohexose isomerase	Isomerisation		Fructose 6-phosphate	Reversible
	Fructose 6-phosphate	Phosphofruktokinase-1	Phosphorylation	ATP	Fructose 1,6-bisphosphate	Irreversible
	Fructose 1,6-bisphosphate	Aldolase	Hydrolysis	–	Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate	Reversible
	Dihydroxyacetone phosphate	Triose phosphate isomerase	Isomerisation	–	Second molecule of glyceraldehyde 3-phosphate	Reversible
Payoff phase	2x glyceraldehyde 3-phosphate	Glyceraldehyde 3-phosphate dehydrogenase	Oxidation and phosphorylation	2 Organic phosphate (Pi)	1,3- Bisphosphoglycerate	Reversible
	1,3- Bisphosphoglycerate	Phosphoglycerate kinase	Releases energy (releases ATP)	ADP 2	3 phosphoglycerate	Reversible
	3 phosphoglycerate	Phosphoglycerate mutase	Releases energy (releases ATP)	–	2-phosphoglycerate	Reversible
	2-phosphoglycerate	Enolase	Releases energy (releases ATP)	Release 2 molecules of water	Phosphoenol pyruvate (PEP)	Reversible
	Phosphoenol pyruvate (PEP)	Pyruvate kinase	Releases energy (releases ATP)	ADP 2	Pyruvate	Irreversible

• But, 3 reactions cannot take place in reverse as they have a large negative free energy changes. They are the reactions which hexokinase, phosphofruktokinase and pyruvate kinase catalyse. So, energetically favourable reactions must replace these reactions

Lets look at the reactions unique to gluconeogenesis:

1. pyruvate———- phosphoenol pyruvate (PEP)

• The conversion of pyruvate to phosphoenol pyruvate takes place in 2 steps 
• The entire process of conversion of pyruvate to phosphoenolpyruvate needs 2 enzymes: pyruvate carboxylase and PEP carboxykinase (PEPCK). 
• This reaction requires a lot of free energy input. They get that by converting pyruvate to oxaloacetate (reaction catalysed by pyruvate carboxylase). This reaction requires ATP. This is the high energy intermediate. The next step is to convert oxaloacetate to pyruvate carboxylase (reaction catalysed by PEP carboxykinase). This reaction requires GTP. 
  
  In short, this reaction takes place like this

Substrate	Enzyme	What the enzyme requires	Product
Pyruvate	Pyruvate carboxylase	ATP and HCO3-	Oxaloacetate
Oxaloacetate	PEPCK	GTP	Phosphoenol pyruvate

Why pyruvate carboxylate is important?

• It has biotin, a carbon dioxide carrier. The reaction catalyses itself proceeds in 2 phases: the conversion of ATP to ADP in order to dehydrate the bicarbonate (carboxyphosphate as an intermediate). A biotin bound carboxyl group is activated and can now transfer to another molecule. The second phase is the transfer of carboxyl group to pyruvate to form oxaloacetate in three steps

Why PEPCK is important?

• It decarboxylates/phosphorylates oxaloacetate to form PEP and GDP
• Oxaloacetate and PEP must transport from the mitochondria to the cytosol. PEP can directly be transported while oxaloacetate needs to convert to aspartate or malate

2. Hydrolytic reactions to bypass irreversible glycolytic reactions

• Reactions catalysed by the enzymes phosphofructokinase and hexokinase has to bypass as they are endergonic in nature
• Fructose1,6-bisphosphotase (FBPase) hydrolyses Frusctose 1,6-bisphosphate fructose 6 phosphate (F6P). F6P is isomerised to glucose 6 phosphate (G6P), which then hydrolyses to glucose by glucose 6 phosphatase. This enzyme is present only in liver and kidney

Substrate	Process	Enzyme	Product
Fructose 1,6-bisphosphate (FBP)	Hydrolysed	Fructose 1,6-bisphosphotase (FBPase)	Fructose-6-phosphate (F6P)
Fructose 6 Phosphate (F6P)	Isomerised	–	Glucose 6 phosphate (G6P)
Glucose 6 phosphate (G6P)	Hydrolysed	Glucose 6 phosphatase	Glucose

Although gluconeogenesis is energetically expensive, it is necessary. The overall reaction is 

2Pyruvate+ 4ATP+ 2GTP+ 2NADH+ 2H+ 4H2O———- glucose + 4ADP + 2GDP + 6Pi + 2NAD

• The entire process requires 6 high energy phosphate groups, 4 from ATP and 2 from GTP. In addition, it requires 2 molecules of NADH. This is in contrast to glycolysis where it requires 2 ATP to break down 1 molecule of glucose
• As mentioned earlier, pyruvate is not the only source for gluconeogenesis. Intermediates of citric acid like citrate, isocitrate, alpha ketoglutarate, succinyl CoA, succinate, fumarate and malate are all capable of oxidation to oxaloacetate
• Similarly, the carbon skeleton of most amino acids which are capable of catabolising to pyruvate or intermediates of citric acid cycle and can eventually be converted to glucose. Such amino acids  are glucogenic
• While animals cannot convert acetyl Co-A derived from fatty acids to glucose, plants and microbes can.

Glycogen is crucial for the storage of glucose. The lack of enzymes necessary to break down glycogen to glucose can result in extremely painful muscle cramps upon exertion and are a symptom of an inherited disease called McArdle’s disease. Let’s look at how glycogen is broken down.

Glycogen metabolism:

The breakdown/ metabolism of glycogen is glycogenolysis. The entire process requires three enzymes: Glycogen phosphorylase, Glycogen debranching enzyme and Phosphoglucomutase. Lets look at each one of them closely.

• Glycogen phosphorylase or phosphorylase: it catalyses the phosphorolysis of glycogen i.e. breaks the bond of glycogen by substituting it with a phosphate group. This process yields glucose-1-phosphate (G1P). This is a rate limiting step. Allosteric interactions and covalent modification regulates this step. This enzyme requires a cofactor pyridoxal-5-phosphate (PLP). The intermediate formed upon glycogen phosphorolysis is an oxonium ion
• Glycogen debranching enzyme: it removes the glycogen branches, making more glucose residues accessible by glycogen phosphorylase. It acts as an alpha (1-4)transglycolase i.e it transfers a new alpha (1-4) linked trisaccharide unit from one branch of glycogen to the non reducing branch of another branch. This process makes 3 more units available for phosphorolysis
• Phosphoglucomutase: it converts G1P to G6P (glucose 6 phosphate). The reaction involves the transfer of a phosphoric group from the active phosphoenzyme to G1P forming glucose 1,6-bisphosphate (G1,6P) which then rephosphorylates to G6P. G6P has several fates. It can continue along thr glycolytic pathway or the pentose phosphate pathway

So, the entire process of glycogen metabolism is:

Substrate	Enzyme	What it does	Product
Glycogen	Glycogen debranching enzyme	Transfers a trisaccharide unit from one branch of glycogen to the non reducing portion of another branch	A glycogen molecule with free glucose residues so they are available for the next step
Glycogen from the previous step	Glycogen phosphorylase	Phosphorolysis I.e. breaks the bond in glycogen and substitute it with a phosphate group	Glucose 1 phosphate (G1P) through an oxonium ion intermediate
G1P	Phosphoglucomutase	Transfers a phosphoric group from phosphoenzyme to G1P	G6P