Faculty of Pharmacy B. Pharm. | Semester-3 Biochemistry ...

Biochemistry(13PH0303)Unit-2Topic: Carbohydrate metabolism

Faculty of PharmacyB. Pharm. | Semester-3

Ms. Hiral K. KapuriyaAssistant ProfessorFaculty of Pharmacy, Marwadi University,Rajkot, Gujarat, 360003, India.

Introduction:

• METABOLIC PATHWAYS:• CATABOLIC PATHWAYS Are involved in oxidative breakdown of larger

complexes. They are usually exergonic in nature• ANABOLIC PATHWAYS Are involved in the synthesis of compounds. They are

usually endergonic in nature.

Characteristics of metabolism:

• 1. Metabolic pathways are mostly irreversible

• 2. Every metabolic pathway has a committed first step.

• 3. All metabolic pathways are regulated.

• 4. Metabolic pathways in eukaryotic cells occur in specific cellular locations

GLYCOLYSIS

• Glycolysis comes from a merger of two Greek words: • Glykys = sweet

• Lysis = breakdown/ splitting

• It is also known as Embden-Meyerhof-Parnas pathway or EMP pathway.

Introduction of Glycolysis:

• GLYCOLYSIS: It is the sequence of 10 enzyme-catalyzedreactions that converts glucose into pyruvate with simultaneousproduction on of ATP.

• In this oxidative process, 1mol of glucose is partially oxidised to2 moles of pyruvate.

• This major pathway of glucose metabolism occurs in the cytosolof all cell.

• This unique pathway occurs aerobically as well as anaerobically& doesn’t involve molecular oxygen.

• It also includes formation of Lactate from Pyruvate.

• The glycolytic sequence of reactions differ from species tospecies only in the mechanism of its regulation & in thesubsequent metabolic fate of the pyruvate formed.

• In aerobic organisms, glycolysis is the prelude to Citric acidcycle and ETC.

• Glycolysis is the central pathway for Glucose catabolism.

Summary of Glycolysis:

• A summary of the process of glycolysis cab be written as follows:

• C6H12O6 + 2ADP + 2Pi + 2NAD+ → 2C3H4O3 + 2H2O + 2ATP + 2NADH + 2H+

• In words, the equation is written as:

• Glucose + Adenosine diphosphate + Phosphate + Nicotinamide adenine dinucleotide

↓

• Pyruvate + Water + Adenosine triphosphate + Nicotinamide adenine dinucleotide + Hydrogen ions

Stages of Glycolysis:

• During glycolysis, a single mole of 6-carbon glucose is broken down intotwo moles of 3-carbon pyruvate by a sequence of 10 enzyme-catalyzedsequential reactions. These reactions are grouped under 2 phases, phase Iand II.

• Stage I comprises “preparatory” reactions which are not redox reactionsand do not release energy but instead lead to the production of a criticalintermediate of the pathway.

• Stage I consists of the first five steps of the glycolysis process.

• Similarly, in Stage II, redox reactions occur, energy is conserved in the formof ATP, and two molecules of pyruvate are formed.

• The last five reactions of glycolysis constitute phase II.

Step 1- Phosphorylation of glucose

• In the first step of glycolysis, theglucose is initiated or primed for thesubsequent steps by phosphorylation atthe C6 carbon.

• The process involves the transfer ofphosphate from the ATP to glucoseforming Glucose-6-phosphate in thepresence of the enzyme hexokinase andglucokinase (in animals and microbes).

• This step is also accompanied byconsiderable loss of energy as heat.

Step 2- Isomerization of Glucose-6-phosphate• Glucose 6-phosphate is

reversibly isomerized to fructose6-phosphate by the enzymephosphohexoisomerase/phosphoglucoisomerase.

• This reaction involves a shift ofthe carbonyl oxygen from C1 toC2, thus converting an aldoseinto a ketose.

Step 3- Phosphorylation of fructose-6-phosphate• This step is the second priming

step of glycolysis, where fructose-6-phosphate is converted into fructose-1,6-bisphosphate in the presence of the enzyme phosphofructokinase.

• Like in Step 1, the phosphate is transferred from ATP while some amount of energy is lost in the form of heat as well.

Step 4- Cleavage of fructose 1, 6-diphosphate• This step involves the unique

cleavage of the C-C bond in thefructose 1, 6-bisphosphate.

• The enzyme fructose diphosphatealdolase catalyzes the cleavage offructose 1,6-bisphosphate betweenC3 and C4 resulting in two differenttriose phosphates: glyceraldehyde3-phosphate (an aldose) anddihydroxyacetone phosphate (aketose).

• The remaining steps in glycolysisinvolve three-carbon units, ratherthan six carbon units.

Step 5- Isomerization of dihydroxyacetone phosphate• Glyceraldehyde 3-phosphate can

be readily degraded in the subsequent steps of glycolysis, but dihydroxyacetone phosphate cannot be. Thus, it is isomerized into glyceraldehyde 3-phosphate instead.

• In this step, dihydroxyacetone phosphate is isomerized into glyceraldehyde 3-phosphate in the presence of the enzyme triose phosphate isomerase.

• This reaction completes the first phase of glycolysis.

Step 6- Oxidative Phosphorylation of Glyceraldehyde 3-phosphate• Step 6 is one of the three energy-

conserving or forming steps of glycolysis.

• The glyceraldehyde 3-phosphate isconverted into 1,3-bisphosphoglycerateby the enzyme glyceraldehyde 3-phosphate dehydrogenase(phosphoglyceraldehyde dehydrogenase).

• In this process, NAD+ is reduced tocoenzyme NADH by the H– fromglyceraldehydes 3-phosphate.

• Since two moles of glyceraldehyde 3-phosphate are formed from one mole ofglucose, two NADH are generated in thisstep.

Step 7- Transfer of phosphate from 1, 3-diphosphoglycerate to ADP• This step is the ATP-generating step

of glycolysis.

• It involves the transfer of phosphate group from the 1, 3-bisphosphoglycerate to ADP by the enzyme phosphoglycerate kinase, thus producing ATP and 3-phosphoglycerate.

• Since two moles of 1, 3-bisphosphoglycerate are formed from one mole of glucose, two ATPs are generated in this step.

Step 8- Isomerization of 3-phosphoglycerate

• The 3-phosphoglycerate isconverted into 2-phosphoglycerate due to theshift of phosphoryl group fromC3 to C2, by the enzymephosphoglycerate mutase.

• This is a reversible isomerizationreaction.

Step 9- Dehydration 2-phosphoglycerate

• In this step, the 2-phosphoglycerate is dehydratedby the action of enolase(phosphopyruvate hydratase) tophosphoenolpyruvate.

• This is also an irreversiblereaction where two moles ofwater are lost.

Step 10- Transfer of phosphate from phosphoenolpyruvate• This is the second energy-generating

step of glycolysis.

• Phosphoenolpyruvate is converted into an enol form of pyruvate by the enzyme pyruvate kinase.

• The enol pyruvate, however, rearranges rapidly and non-enzymatically to yield the keto form of pyruvate (i.e. ketopyruvate). The keto form predominates at pH 7.0.

• The enzyme catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, thus forming ATP.

Enrgetics:

• The overall process of glycolysis results in the following events:

1.Glucose is oxidized into pyruvate.

2.NAD+ is reduced to NADH.

3.ADP is phosphorylated into ATP.

Citric acid cycle:

• The citric acid cycle is the central metabolic hub of the cell.

• The Citric Acid Cycle is also known as: – Kreb’s cycle, TCA (tricarboxylicacid) cycle

• It is the final common pathway for the oxidation of fuel molecule suchas amino acids, fatty acids, and carbohydrates.

• In eukaryotes, the reactions of the citric acid cycle take place insidemitochondria, in contrast with those of glycolysis, which take place inthe cytosol.

• The citric acid cycle is a series of reactions that brings aboutcatabolism of acetyl-coA liberating reducing equivalents which uponoxidation through respiratory chain of mitochondria, generate ATP.

• It plays a central role in the breakdown or catabolism of organic fuelmolecules—i.e glucose and some other sugars, fatty acids, and someamino acids.

• Before these rather large molecules can enter the TCA cycle theymust be degraded into a two-carbon compound called acetylcoenzyme A (acetyl CoA).

• Once fed into the TCA cycle, acetyl CoA is converted into carbondioxide and energy.

• Takes place in the matrix of the mitochondria.

• It happens once for every pyruvate molecule in glycolysis.

• Purpose:• Conversion of Acetyl-CoA to CO2

• Generates reducing equivalents (NADH+H+, FADH2) & GTP to be oxidized inthe respiratory chain to generate ATP.

TCA Cycle with regulation:

Step-1:

• Formation of citrate:

• Citrate formed from condensation of acetyl CoA and oxaloacetate

• Addition of acetyl to the keto double bond of OAA = aldol condensation

• Only cycle reaction with C-C bond formation

• No energy of ATP hydrolysis needed

• Synthase is an enzyme that catalyzes addition to a double bond or elimination to form adouble bond without needing ATP hydrolysis

Step-2:

• Isomerization of citrate:

• Isomerization of citrate (3° alcohol) to isocitrate (2° alcohol)

Step-3:

• Isocitrate Dehydrogenase:

• First oxidative decarboxylation of isocitrate to α-ketoglutarate (α-kg)

• Metabolically irreversible reaction

• One of four oxidation-reduction reactions of the cycle

• Also a Non-hydrolytic cleavage reaction (addition or elimination) • Hydrideion from the C-2 of isocitrate is transferred to NAD+ to form NADH

Step-4:

• α-Ketoglutarate Dehydrogenase Complex:

• Second oxidative decarboxylation reaction

• Also a Non-hydrolytic cleavage reaction (addition or elimination) - α-Ketoglutarate converted to Succinyl-CoA

Step-5:• Succinyl-CoA Synthetase (Formation of succinate):

• Free energy in thioester bond of succinyl CoA is conserved as GTP (or ATP in plants, some bacteria)

• Enzyme: Succinyl-CoA Synthetase

• Two forms in higher animals: One prefers ADP the other GDP)

• SUBSTRATE-LEVEL PHOSPHORYLATION = Formation of ATP directly coupled to the reaction (group transfer reaction)

• Only step where ATP (GTP) is formed directly in the TCA cycle

Step-6:• The Succinate Dehydrogenase (SDH) Complex:

• Located on the inner mitochondrial membrane

• Formation of carbon-carbon double bond

• Succinate is oxidized to fumarate, while FAD is reduced to FADH2

Step-7:

• Fumarase:

• Stereospecific trans addition of water to the double bond of fumarate to form L-malate

Step-8:

• Malate Dehydrogenase:

• Regeneration of oxaloacetate from L-malate

• Enzyme: malate dehydrogenase

• Generates NADH

Energetics:

• TCA cycle is an open cycle

• Operates only under aerobic conditions

• This is the Final common pathway of oxidative metabolism

• Two carbon dioxide molecules are released as a waste product of respiration

Significance of TCA cycle: • Complete oxidation of Acetyl CoA

• As provider of energy

• Final common oxidative pathway

• Fat is burned on the wick of carbohydrates

• Excess carbohydrates are converted to Neutral fat

GLUCONEOGENESIS

The synthesis of glucose from non-carbohydrate compounds is known as gluconeogenesis.

Major substrate/precursors : lactate, pyruvate, glycogenic amino acids, propionate & glycerol.

-Takes place in liver; kidney matrix( 1/3rd).

- Occurs in cytosol and some produced in mitochondria.

Importance of Gluconeogenesis

Brain,CNS,

erythrocytes,testesand kidney medulla

dependent on glucose for cont. supply of energy.

Under anaerobic condition, glucose is the only source to supply skeletal

muscles.

Occurs to meet the basal req of the

body for glucose in fasting for even more than a day.

Effectively clears,certain metabolites

produced in the tissues that

accumulates in blood

Reaction of Gluconeoge

Glucose

Cori Cycle

The

involveing

synthesis

cycle

the

of

glucose in liver

from the skeletal

muscle lactate and

glucose

the reuse of

thus

synthesized by the

muscle for energy

purpose is known

as Cori cycle.

GLYCOGEN ME TABOL ISM

✓Glycogen is a storage form of glucose in animals.

✓Stored mostly in liver (6-8%) and muscle (1-2%)

✓Due to muscle mass the quantity of glycogen in muscle = 250g

and liver =75g

✓Stored as granules in the cytosol.

✓Functions : Liver glycogen – maintain the blood glucose level

Muscle glycogen – serves as fuel reserve

GLYCOGENESIS

❑ Synthesis of glycogen from glucose.

❑ Takes place in cytosol.

❑ Requires UTP and ATPbesides glucose.

❑ Steps in synthesis :

1) Synthesis of UDP- glucose

2) Requirement of primer to initiate glycogenesis

3) Glycogen synthesis by glycogen synthase

4) Formation of branches in glycogen

GLYCOGENOLYSIS

❑Degradation of stored glycogen in liver and muscle constitutes

glycogenolysis.

❑ Irreversible pathway takes place in cytosol.

❑ Hormonal effect on glycogen metabolism :

1) Elevated glucagon – increases glycogen degradation

2) Elevated insulin – increases glycogen synthesis

❑ Degraded by breaking majorly α-1,4- and α-1,6-glycosidic bonds.

❑ Steps in glycogenolysis:

1) Action of glycogen phosphorylase

2) Action of debranching enzyme

3) Formation of glucose-6-phosphate and glucose

Hexose Monophosphate Shunt Pathway:

• Hexose Monophosphate Shunt (HMP Shunt) is also called as pentosephosphate pathway or phosphogluconate pathway.

• It is an alternative pathway to glycolysis and TCA cycle.

• HMP shunt is more anabolic, as it is concerned with the biosynthesisof NADPH and pentose.

• Location:• The enzymes for HMP shunt are located in the cytosol of cells.

• HMP shunt pathway is very little active in highly glycolytic tissue, like skeletalmuscle and non-lactating mammary gland, but, it is highly active in the liver,adipose tissue, erythrocytes, adrenal gland, testis and lactating mammarygland.

• Most of these are dependent on the supply of NADPH.

• Reactions:• The reactions of HMP shunt consists of two phases –

• (i) Oxidative phase, and

• (ii) Non-oxidative phase.

Oxidative phase:• In this phase:-

• (i) Glucose 6-phosphate dehydrogenase oxidizes glucose 6-phosphateirreversibly to 6- phosphoconolactone with the help of NADP which isreduced to NADPH.

• (ii) Gluconolactone hydrolase hydrolyzes 6-phosphoconolactoneirreversibly to 6- phosphogluconate.

• (iii) 6-phosphogluconate dehydrogenase next oxidizes 6-phosphogluconate irreversibly to 3- keto-6-phosphogluconatereducing NADP to NADPH.

• The enzyme then decarboxylates 3-keto-6-phosphogluconate toproduce ribulose 5-phosphate.

Non-oxidative phase:-

• The early reaction of this phase isomerizes ribulase to ribose 5-phosphate, which is used in synthesizing nucleotide.

• Later steps convert ribulose and ribose phosphate into fructose 6-phosphate and glyceraldehyde 3-phosphate.

• (i) Phosphopentose isomerase isomerizes ribulose 5-phosphate into ribose5- phosphate.

• (ii) Phosphopentose 3-epimerase at the same time isomerizes ribulose 5-phosphate into xylulose 5-phosphate. -3-

• (iii) Transketolase transfers C2 group from a ketose (xylulose 5-phosphate)to an aldose (ribose 5-phosphate) changing them to glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate.

• (iv) Transaldolase transfer carbon from a ketose (sedoheptulose 7-phosphate to an aldose (glyceraldehyde 3-phosphate), changing them toerythrose 4-phosphate and fructose 6-phoaphate.

• (v) Transketolase transfer carbon of another xylulose 5-phosphate to theerythrose 4- phosphate produced in the reaction, changing them toglyceraldehyde 3-phosphate and fructose 6-phosphate.

Significance:• (i) HMP shunt is a unique pathway, generating important products, like

pentose and NADPH which are needed for the biosynthetic reactions andvarious other functions.

• (ii) This pathway utilizes NADP+ instead of NAD+ and contributes little toATP production, so it is less active in muscles which require more energy.

• (iii) Pathway generates NADPH which is utilized as electron donor insynthesis of fatty acids, steroids, cholesterol etc. so it is more active in liver,testis, adipocytes etc.

• (iv) The rate of this pathway increased in leucocytes during phagocytosis,NADPH generated is utilized by NADPH oxidase in producing superoxideradicals for destroying phagocytized materials.

• (v) In erythrocytes, NADPH generated by this pathway is utilized byglutathione reductase in reducing oxidized glutathione.

G6PD Deficiency

• It is the most common red cellenzymopathy associated withhemolysis.

Symptoms

• Persons with this condition do not display any signs of thedisease until their red blood cells are exposed to certainchemicals in food or medicine, or to stress.

• Symptoms are more common in men and may include:• Dark urine

• Enlarged spleen

• Fatigue

• Pallor

• Rapid heart rate

• Shortness of breath

• Yellow skin color

TYPE ENZYME DEFECT CLINICAL FEATURES

Type I (Von Gierke’s

disease)

Glucose-6-

phosphatase

deficiency.

Glycogen accumulates in hepatocytes and

renal cells, enlarged liver and kidney, fasting

hypoglycemia, lactic acidemia; hyperlipidemia;

ketosis; gouty arthritis.

Type II (Pompe’s

disease)

Acid maltase

deficiency

Diminished muscle tone, heart failure, enlarged

Tongue

Type III (Cori’s

disease, Forbe disease)

Debranching enzyme

deficiency

Hypoglycemia, enlarged liver, cirrhosis, muscle

weakness, cardiac involvement

Type IV (Andersen’s

disease)

Branching enzyme

deficiency

Enlarged liver & spleen, cirrhosis, diminished

muscle tone, possible nervous system

involvement

Type V (Mcardle’s

disease)

Muscle phosphorylase

deficiency

Muscle weakness, fatigue and muscle cramps

GLYCOGEN STORAGE DISEASES

Hormonal regulation of blood glucose level and Diabetes mellitus

• Insulin and glucagon are hormones secreted byislet cells within the pancreas. They are bothsecreted in response to blood sugar levels, butin opposite fashion.

• Insulin is normally secreted by the beta cells (atype of islet cell) of the pancreas. The stimulusfor insulin secretion is a HIGH blood glucose.

• Although there is always a low level of insulinsecreted by the pancreas, the amount secretedinto the blood increases as the blood glucoserises.

• Similarly, as blood glucose falls, the amount ofinsulin secreted by the pancreatic islets goesdown.

Role of Insulin:

• Insulin has an effect on anumber of cells, includingmuscle, red blood cells, and fatcells.

• In response to insulin, these cellsabsorb glucose out of the blood,having the net effect of loweringthe high blood glucose levelsinto the normal range.

The Role of Glucagon in Blood Glucose Control

• The effect of glucagon is to makethe liver release the glucose ithas stored in its cells into thebloodstream, with the net effectof increasing blood glucose.

• Glucagon also induces the liver(and some other cells such asmuscle) to make glucose out ofbuilding blocks obtained fromother nutrients found in thebody (eg, protein).

Regulation:

Somatostatin• Somatostatin (also known as growth hormone-inhibiting

hormone (GHIH) or somatotropin release-inhibitingfactor(SRIF)) or somatotropin release-inhibiting hormone

• It is a peptide hormone that regulates the endocrine system andaffects neurotransmission and cell proliferation via interaction with Gprotein-coupled somatostatin receptors.

• Somatostatin inhibits insulin and glucagon secretion.

• Metabolic Effect:• Suppresses glucagon release from α cells (acts locally);• Suppresses release of Insulin, Pituitary tropic hormones, gastrin and secretin.

• ACTION OF SOMATOSTATIN:• Somatostatin DECREASE glucagon secretion & Lowers the blood glucose level

Symptoms:

Effect of other enzymes on blood glucose level

URONIC ACID PATHWAY

• Alternative oxidative pathway for glucose.

• Synthesis of glucorinc acid, pentoses and vitamin (ascorbic acid).

• Normal carbohydrate metabolism, phosphate esters are involved –but in uronic acid pathway free sugars and sugar acids are involved.

• Steps of reactions :• Formation of UDP-glucoronate• Conversion of UDP- glucoronate to L-gulonate• Synthesis of ascorbic acid in some animals• Oxidation of L-gulconate