Glycolysis Dr.S.Chakravarty MD

A year and a half old Amish girl from Pennsylvania girl is being seen by the hematologist after her pediatrician found her to be severely anemic with splenomegaly and jaundice. Her mother gives a possible history of a “blood problem” in her family but doesn’t know for sure.

 Her hemoglobin electrophoresis was normal, and the complete

blood count (CBC) revealed a normocytic anemia. The platelet and white blood cell counts are normal. On the peripheral smear, there are many bizarre erythrocytes including spiculated cells. Heinz bodies are absent.

Questions:-

What can be the diagnosis?

What is the biochemical basis of the clinical features?

Learning objectives:

Analyze the importance of Glycolytic pathway that it can produce ATPs in both aerobic and anaerobic environment

Differentiate between substrate level and oxidative phosphorylation

List the GLUT transporters and classify them based on insulin dependency

List the rate limiting and irreversible steps of Glycolysis and their regulation

Explain the Importance of Embden Meyerhof pathway

Describe the clinical features of pyruvate kinase deficiency

Calculate the Energy generated during aerobic and anaerobic Glycolysis

Metabolism: Defined as sum of all chemical changes that occur in

the body

Divided into two groups :

1. Anabolism : synthesis of complex molecules from simple molecules like glucose to glycogen.

2. Catabolism : breakdown of complex molecules like proteins, carbohydrates and lipids to simple molecules such as CO2, H2O and NH2

Three stages of catabolism : Carbohydrates Lipids Proteins

Monosaccharides Fatty acids Glycerol

Aminoacids

Acetyl Co-A

TCA cycle

CO2 + H2O + ATP

Glucose uptake by cells:Major Glucose transporters (GLUT):Receptor Tissues Km Function Facilitative bidirectional transporters GLUT – 1 Most tissues (Brain,

RBCs, Colon ,Placenta)1 mMLOW Km-High affinity Basal uptake

GLUT – 2 LiverPancreas Small intestineKidney

15 mMHIGH Km(Low affinity transporter)

• Uptake and release of glucose by liver( AFTER A MEAL )

• Glucose sensor GLUT-3 Brain

KidneyPlacenta

1 mMLow Km-High

AFFINITYBasal uptake

GLUT – 4 Skeletal muscle

Adipose tissue Heart

5 mM • Insulin stimulated glucose uptake

GLUT -5 Small Intestine •Absorption

Sodium dependent unidirectional transporter

SGLT1 Small Intestine and Kidney

•Active uptake of glucose against a concentration gradient

NORMAL BLOOD GLUCOSE CONCENTRATION 4-6 mM (70-110 mg/dl)Glut 1 and Glut 3 are at Vmax at Normal glucose concentrationRECALL :Km is inversely proportional to affinity

Salient features of Glycolysis:Occurs in the cytoplasm of all the cells in the body

Immediate /basal source of energy (ATP) is provided by this pathway.

It provides intermediates for other pathways like Pyruvate, glucose-6-PO4, and Dihydroxyacetone phosphate etc.

Hub of carbohydrate metabolism – all carbs are finally converted to glucose or intermediates of Glycolysis before being metabolized.

ALL CELLS CARRY OUT GLYCOLYSIS Glycolysis is the ONLY source of ATPs in:

Cornea and lens of the eyeRenal medullaRBCsSkin Cancerous cells.

Two types of Glycolysis:A. Aerobic Glycolysis : formation of Pyruvate as end

product with production of ATP and NADH when oxygen is available

B. Anaerobic Glycolysis : formation of lactate as end product with production of only ATP in the absence of oxygen .

Allows continuous production of ATPs in cells without mitochondria or cells deprived of oxygen

Glycolysis

Glucose

Glucose -6-PO4

Fructose -6-PO4

Fructose -1,6-bisphosphate

Glucokinase /Hexokinase

Phosphofructokinase-1

ATP

ADP

ATP

ADP

Energy consuming phase

Irreversible step -1

Irreversible step -2

Rate limiting step

Phosphohexose isomerase

Glycolysis, Gluconeogenesis, The HMP shunt ,GlycogenesisGlycogenolysis

Reversible but driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis.

GlycolysisSplitting phase – into molecules of 3 carbons each

Fructose -1,6-bisphosphate

Glyceraldehyde-3-PO4 Dihydroxyacetone phosphate

Aldolase A

6C

3C 3CIsomerase

Glycerol -3-po4

Glycerol -3-po4 dehydrogenase

Fatty acid synthesis

Energy yielding phase

Glyceraldehyde-3-PO4

1,3 bis phosphoglycerate

NAD

NADH

Glyceraldehyde-3-PO4

dehydrogenase

3-phosphoglycerate

2-phosphoglycerate

Phosphoenolpyruvate

Pyruvate

ADP

ATPPyruvate Kinase Irreversible step

-3

Pathway repeats twice because of 2 molecules of Glyceraldehye 3-PO4 formed

ADP

ATPPhosphoglycera

te kinase

Enolase (-) Fluoride

Substrate level phosphorylation

Phosphoglycerate mutase

Energy yield from one molecule of glucose

ATPs consumed during Glycolysis

1 – Glucokinase 1 – Phosphofructokinase

ATPs produced during Glycolysis

2 – Phosphoglycerate kinase 2 – Pyruvate kinase

NADH produced Glycolysis (Aerobic pathway / or cells with mitochondria)

2 – Glyceraldehyde-3-PO4 dehydrogenase (NADH = 2.5 ATPs)

Net gain in ATPs during Aerobic glycolysis = (4 + 5 – 2 = 7 ATPs)

Regeneration of NAD+

Very little NAD in the cytosol.

NADH NAD+ + 2 electrons

In Aerobic tissues: by transferring the electrons to mitochondria to produce ATP by shuttle mechanisms.

In Anaerobic tissues or aerobic tissues devoid of oxygen: by producing lactic acid.

Anerobic glycolysis:

Pyruvate

Lactate

Lactate Dehydrogenase

NADH

NADH

NAD

NAD

• Net energy gain during anaerobic Glycolysis is only 2 ATPs• NADH produced during anaerobic Glycolysis is utilized during lactate dehydrogenase step

Glycolysis in Erythrocytes:

1,3 Bis phosphoglycerate

3-phosphoglycerate

2,3 Bis phosphoglycerate(2,3BPG)

Mutase

Phosphatase

Phosphoglycerate kinase

ADP

ATP

• Net ATP production during production of 2,3 BPG in RBCs = 0 ATPs• Increase in 2,3 BPG shifts the oxygen dissociation curve to the right

Regulation of Glycolysis:

Regulation at the level of Glucokinase/Hexokinase

Regulation at Phosphofructokinase

Regulation of Pyruvate kinase

Hormonal regulation (mainly liver): Insulin favors Glycolysis and Glucagon inhibits Glycolysis

Difference between Hexokinase and Glucokinase Hexokinase Glucokinase

Substrate specificity All hexoses Mainly Glucose

Km Low (high affinity)Works at normal glucose concentration

High (low affinity) works only when glucose levels are elevated

Location Universal Mainly liver and Beta cells of pancreas

Vmax (rate of reaction) Low High

Glucose-6-PO4 (Allosteric inhibition)

Inhibits the enzyme No inhibition

Insulin No regulation Positive regulation

Diabetes Mellitus :

Insulin dependent Diabetes Mellitus (IDDM) – def of insulin due to autoantibodies against Beta cells

Non insulin dependent Diabetes mellitus (NIDDM) – insulin receptor resistance

Maturity onset diabetes of the young – (MODY) – mutation in the Glucokinase gene.

Allosteric Regulation of PFK-1:

Situation of high energy levels in the cells indicated by:

1. High ATP:2. High citrate levels :

Situation of low energy in the cells indicated by:

1. High ADP /AMP level2. High fructose 2,6 bisphosphate

Allosteric inhibition of PFK-1

Allosteric activation

Fructose -6-po4

Fructose -1,6- Bisphosphate

Fructose -2,6- Bisphosphate

PFK-1

PFK-2

Insulin

Glucagon

Regulation of PFK -1 :

Covalent modification of Pyruvate kinase :

Pyruvate Kinase

Pyruvate Kinase

po4ATP ADP

Protein kinase A

Glucagon cAMP(+)

(+)

Inactive

Active

(+)

Protein phosphatase Insulin

(+)

Inhibition of Glycolysis in liver and increase blood glucose

Pyruvate kinase def : in RBCs

Second most common cause for enzyme deficiency related hemolytic anemia.

Def causes decreased ATP production in RBCs

Decreased energy to fuel the pumps required to maintain the biconcave, flexible shape of RBCs.

Red cell damage and phagocytosis – premature death and lysis – hemolytic anemia (chronic hemolysis)

Absence of Heinz bodies ( to differentiate G6PD def)

Under conditions of anaerobic glycolysis, the NAD+ required by glyceraldehyde-3-phosphate dehydrogenase is supplied by a reaction catalyzed by which of the following enzymes?

Glycerol-3-phosphate dehydrogenaseAlpha-ketoglutarate dehydrogenaseLactate dehydrogenaseMalate dehydrogenasePDH

After consumption of a carbohydrate-rich meal, the liver continues to convert glucose to glucose-6-phosphate. The liver’s ability to continue this processing of high levels of glucose is important in minimizing increases in blood glucose after eating. What is the best explanation for the liver’s ability to continue this conversion after eating a carbohydrate-rich meal?

The Hepatocyte cell membrane’s permeability for glucose-6-phosphate

The high maximum reaction rate (high Vmax) of Glucokinase

The inhibition of Glucokinase by high glucose-6-phosphate

The lack of Glucokinase level regulation by insulinThe low Michaelis-Menten (Km) constant of Glucokinase

Fates of Pyruvate:

Various fates of Pyruvate:

How many ATPs are produced from oxidation of 2 molecules of Glucose ?

A. 32B. 38C. 64D. 48E. 0

Which of the following best describes the effect of ATP on PFK 1 ?

Thank You