GLYCOLYSISGLYCOLYSIS

Dr Vivek Joshi,MDFall 2015

Learning ObjectivesLearning ObjectivesIntroduction Bio Medical ImportanceSiteReactions of the pathwayEnergetic of Glycolysis Regulation of the pathwayClinical Significance

IntroductionIntroduction

Glycolysis was one of the first metabolic pathways Glycolysis was one of the first metabolic pathways studied and is also one of the best understood studied and is also one of the best understood

Also known as the Embden-Meyerhof pathway

IntroductionIntroduction Major pathway for oxidation of Glucose Glucose Pyruvate/Lactate Glycolysis: Aerobic /Anaerobic

Aerobic –Glucose Pyruvate

Anaerobic Glucose Pyruvate Lactate

NAD+NADH + H+

ETC-ATP

NADH + H+NAD+

Bio Medical ImportanceBio Medical Importance

Provision of energy Anaerobic Glycolysis-Skeletal muscle during

exercise Aerobic Glycolysis- Heart muscles -Poor survival

under ischemic conditions Hemolytic anemia- Pyruvate kinase deficiency

Sites of GlycolysisSites of Glycolysis

Sub cellular site : In the cytoplasm of eukaryotic Cells

Tissue site :Occurs in practically all living cellsAerobic glycolysis

In the presence of oxygen In the Cells with mitochondria

Anaerobic glycolysis In the absence of oxygen In the RBCs & contracting skeletal muscle

Reactions of the pathwayReactions of the pathway

11ststIrreversible Step Irreversible Step Glucose to Glucose 6-phosphate by Glucose to Glucose 6-phosphate by Hexokinase/GlucokinaseHexokinase/Glucokinase

-Nonionic glucose is converted into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugar.-Biologically inert glucose becomes activated into a labile form capable of being further metabolized

Hexokinase v/s Glucokinase Hexokinase v/s Glucokinase Uptake of Glucose and phosphorylation

Uptake of Glucose by the tissue-Glucose transporters(GLUT)

Phosphorylation of Glucose Hexokinase

Wide tissue distributionLow Km (High affinity for glucose)Low VmaxInhibited by Glucose-6-PUtilization of glucose to provide energy even

when [glucose] is low

Glucokinase Liver & islet cells of pancreasHigh Km- Low affinity for the substrateHigh Vmax -Can effectively phosphorylate Glucose in the cell. Induced by InsulinAt high blood glucose levels (After meals)-Glucose uptake by GLUT 2 in the liver and pancreas with phosphorylation by GlucokinaseActs as ‘glucose sensor’ in islets cells of pancreas–determines threshold for insulin secretion

Hexokinase v/s GlucokinaseHexokinase v/s Glucokinase

Conversion of Glucose 6-phosphate Conversion of Glucose 6-phosphate to Fructose 6-phosphateto Fructose 6-phosphate

22ndnd Irreversible step Irreversible step- - Fructose 6 phosphate to Fructose 6 phosphate to Fructose 1,6 bisphosphate by Fructose 1,6 bisphosphate by Phosphofructokinase -1Phosphofructokinase -1

PFK-1 is the most regulated enzyme.

Cleavage of (6C) Cleavage of (6C) Fructose 1,6 biphosphate Fructose 1,6 biphosphate to 2 to 2 triose sugarstriose sugars

Phosphotriose Isomerase Phosphotriose Isomerase converts dihydroxyacetone converts dihydroxyacetone phosphate to Glyceraldehyde 3-phosphatephosphate to Glyceraldehyde 3-phosphate

Glyceraldehyde 3-phosphate Dehydrogenase Glyceraldehyde 3-phosphate Dehydrogenase catalyzes the only step where NADH is synthesizedcatalyzes the only step where NADH is synthesized

Phosphoglycerate kinasePhosphoglycerate kinase catalyzes the catalyzes the first first substrate-level phosphorylationsubstrate-level phosphorylation step in glycolysis step in glycolysis

A mutase moves the phosphate from a 3- to a 2- position on glycerate

EnolaseEnolase results in generation of the high-energy results in generation of the high-energy bond in Phosphoenolpyruvatebond in Phosphoenolpyruvate

EnolaseEnolase is inhibited by is inhibited by FluorideFluoride (used in blood (used in blood collection tubes for collection tubes for blood glucose estimationsblood glucose estimations))

Pyruvate kinase Pyruvate kinase catalyzes the catalyzes the second substrate-second substrate-level phosphorylationlevel phosphorylation step in glycolysis step in glycolysis

GlucoseGlucose

PYRUVATEPYRUVATE

ACETYL Co A ACETYL Co A OXALOACETATEOXALOACETATE

CITRIC ACID CYCLE (CITRIC ACID CYCLE (TCATCA)) GLUCONEOGENESIS GLUCONEOGENESIS

Aerobic GlycolysisLDH

Alanine

Transaminase

Pyruvate Carboxylase

(BIOTIN)

Pyruvate Dehydrogen

ase ATP

ADP

CO2

CO 2

NAD +NADH +

H+

LactateCoASH

Metabolic Fate of Pyruvate

Aerobic conditions (Aerobic conditions (Presence of oxygenPresence of oxygen)) PyruvatePyruvate can be converted to can be converted to acetyl-CoAacetyl-CoA and enter the and enter the

Krebs CycleKrebs Cycle Acetyl-CoAAcetyl-CoA is completely oxidized is completely oxidized during Krebs cycle during Krebs cycle

and generates ATPand generates ATP through oxidative phosphorylation through oxidative phosphorylation Anerobic conditions (Anerobic conditions (Absence of oxygenAbsence of oxygen)/Absence )/Absence

of mitochondriaof mitochondria Seen mainly Seen mainly in RBCs and the contracting skeletal in RBCs and the contracting skeletal musclemuscle PyruvatePyruvate is converted to is converted to lactatelactate

Metabolic Fate of Pyruvate

ConversionConversion of of PyruvatePyruvate to to LactateLactate by by Lactate Dehydrogenase (LDH)Lactate Dehydrogenase (LDH)

PyruvatePyruvate LactateLactate

NADH + H+ NAD+

Lactate Dehydrogenase (LDH)

Lactate is the final product of anerobic glycolysis

Is formed mainly in the RBC, lens and cornea of the eye, kidney medulla, testes and leukocytes

Is also formed in the intensely exercising skeletal muscle ( accumulation causes cramps)

Energy yield of GlycolysisEnergy yield of Glycolysis

Aerobic GlycolysisAerobic Glycolysis ((Glucose to pyruvateGlucose to pyruvate) ) == 8 ATP/mol o8 ATP/mol of glucosef glucoseGlucose + 2 Pi + 2 ADP + 2 NAD Glucose + 2 Pi + 2 ADP + 2 NAD 2 Pyruvate + 2 ATP + 2 NADH 2 Pyruvate + 2 ATP + 2 NADH

Anaerobic Glycolysis Anaerobic Glycolysis ((Glucose to lactateGlucose to lactate) ) ==2 ATP/mol 2 ATP/mol of glucoseof glucoseGlucose + 2 Pi + 2 ADP 2 Lactate + 2 ATP Glucose + 2 Pi + 2 ADP 2 Lactate + 2 ATP

Irreversible and Regulated steps catalyzed by: Glucokinase- Induction and Repression Hexokinase- Product Inhibition Phosphofructokinase-1- Allosteric Regulation

( Most regulated enzyme) Pyruvate Kinase- Covalent Modification

Regulation of GlycolysisRegulation of Glycolysis

PFK-2 (Controls PFK-1 of Glycolysis in the liver)-Covalent Modification

Product Inhibition –Inhibited by Glucose -6-P(Product)HEXOKINASEHEXOKINASE

Induced by Insulin Near complete Deficiency-Neonatal Type I

Diabetes Mutation of Glucokinase gene

Decrease Km- Hyperinsulinemia and Hypoglycemia

Increase Km-Maturity Onset Diabetes of the Young(MODY)

GLUCOKINASEGLUCOKINASE

Most Regulated Enzyme Positive Allosteric Effector- High Fructose -

2,6 bis phosphate (Liver) High ADP (Liver)/ High AMP(Muscle)

Negative Allosteric Effector –Citrate (Liver)

Phosphofructokinase-1(PFK-1)

F2,6 BP

Phosphofructokinase-1(PFK-1)Potent activator -Fructose 2,6 bisphosphate Fructose 2,6 bisphosphate (F2,6BP) (F2,6BP)

Fructose-6 Fructose-6 Fructose-1,6 BP Fructose-1,6 BP PFK-1PFK-1

++

Fructose-6 Fructose-6 Fructose-2,6 BP Fructose-2,6 BP PFK-2PFK-2

Fructose 2,6 bisphosphate Fructose 2,6 bisphosphate (F2,6BP) (F2,6BP)

Synthesis of F2,6BP is catalyzed by the bifunctional enzymePhosphofructokinase-2/fructose-2,6-bisphosphatase

(PFK-2/F-2,6-BPase)

Phosphofructokinase-2(PFK-2)

The only example of a citric acid cycle

intermediate regulating a

glycolytic enzyme

Phosphofructokinase-1(PFK-1)Inhibitors-

Citrate and ATP Citrate and ATP

Pyruvate Kinase

Glucagon

- Pyruvate Kinase-P (Inactive)

-PFK-2-P (Inactive)

Pyruvate Kinase

Pyruvate Kinase

(Active)

Pyruvate Kinase

(Inactive)

GLYCOLYSIS IS INHIBITED

Pyruvate Kinase

Glucagon/Epinephrine-Pyruvate Kinase-P (Inactive)

-PFK-2-P (Inactive)

F26BPaseinactive

PFK-2Inactive

F26BPaseactive

PFK-2

Active

P

P

Active Protein kinase A

High Glucagon (cAMP) as in hypoglycemia

Decreased

GLYCOLYSIS

ATP ADP

F 2,6BP

F 6BP

High Glucagon (High Glucagon (cAMPcAMP) –) –PFK-2 PFK-2 InactiveInactive………………

Fructose-2,6-bisphosphateFructose-2,6-bisphosphate thus thus decreases in liver decreases in liver cells in response cells in response to a glucagon-activated cAMP to a glucagon-activated cAMP signal cascadesignal cascade. Downstream effects include:. Downstream effects include:

Glycolysis slows because Glycolysis slows because of the decreased of the decreased

concentration of fructose-2,6-bisphosphateconcentration of fructose-2,6-bisphosphate

Gluconeogenesis Increases becauseGluconeogenesis Increases because of the of the

decreased concentration of fructose-2,6-decreased concentration of fructose-2,6-

bisphosphatebisphosphate

Well fed state/Carbohydrate rich diet /High Insulin- Well fed state/Carbohydrate rich diet /High Insulin- INCREASEINCREASE in the : in the : Uptake of Glucose by GLUT 4 (Uptake of Glucose by GLUT 4 (Skeletal muscle and adipose tissue)Skeletal muscle and adipose tissue) Utilization of glucose by oxidationUtilization of glucose by oxidation Anabolic pathwaysAnabolic pathways

Hypoglycemia/Fasting /Starvation/ High Glucagon/Epinephrine-Hypoglycemia/Fasting /Starvation/ High Glucagon/Epinephrine-DECREASEDECREASE in the : in the : Utilization of glucose by oxidation by Utilization of glucose by oxidation by Inactivation/Repression of the Inactivation/Repression of the

key Glycolytic enzymeskey Glycolytic enzymes

Hormonal Control over Hormonal Control over GlycolysisGlycolysis

Clinical SignificanceClinical Significance

Enzyme deficiencies -Pyruvate kinase DeficiencyLactic AcidosisCancer cells2,3,BisphosphoglycerateFluoride and blood glucose estimations

Pyruvate kinase deficiencyPyruvate kinase deficiency Genetic defect Genetic defect associated with mutant enzyme-A severe associated with mutant enzyme-A severe

decrease in Pyruvate kinase activity decrease in Pyruvate kinase activity and rate of and rate of glycolysisglycolysis Affects mainly RBCs Affects mainly RBCs that depend solely on anaerobic that depend solely on anaerobic

glycolysis for energy and maintaining structural integrityglycolysis for energy and maintaining structural integrity Associated with the Associated with the rise in 2,3-BPG rise in 2,3-BPG levels-Shift of levels-Shift of ODC to ODC to

the rightthe right Clinical presentation: Clinical presentation:

# # Hemolytic Anemia Hemolytic Anemia due to lysis of RBCs; usuallydue to lysis of RBCs; usually associated with precipitating factors (oxidant stress,associated with precipitating factors (oxidant stress, infection)infection)

##DiagnosisDiagnosis: Levels of pyruvate kinase activity in RBCs: Levels of pyruvate kinase activity in RBCs##TreatmentTreatment: Usually no therapy required: Usually no therapy required

Lactic AcidosisLactic Acidosis

Accumulation of lactic acid in bloodAccumulation of lactic acid in blood As a result of As a result of failure of delivery of oxygenfailure of delivery of oxygen to tissues –to tissues –

Dependent on Dependent on anerobic glycolysisanerobic glycolysis May be seen associated with:May be seen associated with:

Severe myocardial infarctionSevere myocardial infarctionCardiac muscles-Adapted for aerobic GlycolysisCardiac muscles-Adapted for aerobic GlycolysisPoor survival under ischemic conditionsPoor survival under ischemic conditions

Uncontrolled hemorrhageUncontrolled hemorrhage

Cancer cellsCancer cells

Very Very rapid glucose uptake rapid glucose uptake (shown in 1928 by the German (shown in 1928 by the German Biochemist, Otto Warburg)Biochemist, Otto Warburg)

Commonly experience hypoxia Commonly experience hypoxia and depend on and depend on anaerobic anaerobic glycolysisglycolysis for most of their ATPfor most of their ATP

Have a very Have a very HIGH glycolytic rateHIGH glycolytic rate (lesser mitochondria)(lesser mitochondria)

Mature Erythrocytes Mature Erythrocytes --Dependent on Glucose as fuelDependent on Glucose as fuelGlucose oxidized by Glucose oxidized by Anaerobic GlycolysisAnaerobic Glycolysis(Lack (Lack

MitochondriaMitochondria)) for Energy ,maintain ion channel and for Energy ,maintain ion channel and structural integrity structural integrity

Enzyme Enzyme Bisphosphoglycerate mutase Bisphosphoglycerate mutase Synthesize Synthesize 2,3-2,3-BisphosphoglycerateBisphosphoglycerate is synthesized from an intermediate is synthesized from an intermediate of glycolysis, 1,3 Bisphosphoglycerateof glycolysis, 1,3 Bisphosphoglycerate

The 2,3-Bisphosphoglycerate Pathway in The 2,3-Bisphosphoglycerate Pathway in ErythrocytesErythrocytes

The 2,3-Bisphosphoglycerate The 2,3-Bisphosphoglycerate Pathway in ErythrocytesPathway in Erythrocytes

2,3-BPG in the RBC’s2,3-BPG in the RBC’s Stabilises the Taut formStabilises the Taut form/ “T” /deoxy form of Hb/ “T” /deoxy form of Hb Favours unloading Favours unloading of Oxygen to the tissuesof Oxygen to the tissues High BPG content High BPG content shifts Oxygen dissociation curve to shifts Oxygen dissociation curve to

the rightthe right High Altitude High Altitude –Increased 2,3-BPG–Increased 2,3-BPG Fetus-Fetus-Inability of 2,3-BPG to bind to fetal Hb (Inability of 2,3-BPG to bind to fetal Hb ( chains chains))

RECALL…………………..RECALL…………………..

Fluoride and Blood Glucose estimationsFluoride and Blood Glucose estimations

Blood glucose concentration Blood glucose concentration is used for the diagnosis of is used for the diagnosis of DiabetesDiabetes

Fluoride bulb Fluoride bulb is used for blood glucose estimation - is used for blood glucose estimation - Mixture of Mixture of anticoagulant with Sodium Fluoride anticoagulant with Sodium Fluoride ((Enzyme inhibitorEnzyme inhibitor))

FluorideFluoride (used as sodium fluoride) is a strong (used as sodium fluoride) is a strong competitive competitive inhibitor of Enolaseinhibitor of Enolase,, blocking glycolysis. blocking glycolysis.

Addition of Enzyme inhibitor to the Addition of Enzyme inhibitor to the blood inhibits blood inhibits Glucose utilisation in vitro .Glucose utilisation in vitro .

Prevents false low values Prevents false low values for blood glucosefor blood glucose

Thank YouThank You