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
Monosaccharides
Lipids
Fatty acids
Glycerol
Proteins
Aminoacids
Acetyl Co-A
CO2 + H2O + ATP
TCA cycle
Glucose uptake by cells:Major Glucose transporters (GLUT):
Receptor
Tissues
Km
Function
Facilitative bidirectional transporters
GLUT – 1
Most tissues (Brain, RBCs,
Colon ,Placenta)
1 mM
LOW Km-High affinity
Basal uptake
GLUT – 2
Liver
Pancreas
Small intestine
Kidney
15 mM
HIGH Km
(Low affinity transporter)
• Uptake and release of glucose by
liver( AFTER A MEAL )
GLUT-3
Brain
Kidney
Placenta
1
mM
Low Km-High AFFINITY
Basal uptake
GLUT – 4
Skeletal muscle
5 mM
• Insulin stimulated
• Glucose sensor
Adipose tissue
Heart
GLUT -5
Small Intestine
glucose uptake
•Absorption
Sodium dependent unidirectional transporter
SGLT1
Small Intestine and Kidney
NORMAL BLOOD GLUCOSE CONCENTRATION 4-6 mM (70-110 mg/dl)
Glut 1 and Glut 3 are at Vmax at Normal glucose concentration
RECALL :Km is inversely proportional to affinity
•Active uptake of glucose against a
concentration gradient
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 eye
 Renal medulla
 RBCs
 Skin
 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
Energy consuming phase
ATP
Irreversible step -1
Glucokinase
/Hexokinase
Glycolysis,
Gluconeogenesis,
The HMP shunt ,
Glycogenesis
Glycogenolysis
ADP
Glucose -6-PO4
Reversible but driven forward because of a low
concentration of F6P, which is constantly consumed
during the next step of glycolysis.
Phosphohexose
isomerase
Fructose -6-PO4
Rate limiting step
ATP
Irreversible step -2
Phosphofructokinase-1
ADP
Fructose -1,6-bisphosphate
Glycolysis
Splitting phase – into molecules of 3 carbons each
Fructose -1,6-bisphosphate
Aldolase A
6C
Fatty acid synthesis
Glycerol -3-po4
Glycerol -3-po4
dehydrogenase
Glyceraldehyde-3-PO4
3C
Isomerase
Dihydroxyacetone
phosphate
3C
Glyceraldehyde-3-PO4
NAD
Energy yielding phase
Glyceraldehyde-3PO4 dehydrogenase
NADH
1,3 bis phosphoglycerate
ADP
Substrate level
phosphorylation
Phosphoglycerate
kinase
ATP
3-phosphoglycerate
Phosphoglycerate
mutase
2-phosphoglycerate
Enolase
(-) Fluoride
Phosphoenolpyruvate
ADP
Irreversible step -3
Pyruvate Kinase
ATP
Pyruvate
Pathway
repeats twice
because of 2
molecules of
Glyceraldehye
3-PO4
formed
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
NADH
NADH
Lactate
Dehydrogenase
NAD
NAD
Lactate
• 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
Mutase
ADP
2,3 Bis phosphoglycerate
(2,3BPG)
Phosphoglycerate
kinase
ATP
Phosphatase
3-phosphoglycerate
• 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
All hexoses
Mainly Glucose
Low (high affinity)
High (low affinity)
Works at normal glucose
concentration
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
Substrate specificity
Km
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 :

Allosteric
inhibition of
PFK-1
Situation of low energy in the cells indicated by:
1. High ADP /AMP level
2. High fructose 2,6 bisphosphate
Allosteric
activation
Insulin
Fructose -6-po4
PFK-2
PFK-1
Glucagon
Fructose -2,6Bisphosphate
Fructose -1,6Bisphosphate
Regulation of PFK -1 :
Covalent modification of Pyruvate kinase :
Glucagon
cAMP
(+)
Protein kinase A
Inhibition of Glycolysis in liver
and increase blood glucose
(+)
ATP
po4
ADP
Pyruvate Kinase
Pyruvate Kinase
Active
Inactive
(+)
Protein phosphatase
(+)
Insulin
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-3phosphate dehydrogenase is supplied by a
reaction catalyzed by which of the
following enzymes?
 Glycerol-3-phosphate dehydrogenase
 Alpha-ketoglutarate dehydrogenase
 Lactate dehydrogenase
 Malate dehydrogenase
 PDH
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 insulin
The low Michaelis-Menten (Km) constant of Glucokinase
Various fates of Pyruvate:
 How many ATPs are produced from oxidation of 2





molecules of Glucose ?
A. 32
B. 38
C. 64
D. 48
E. 0
Which of the following best describes the effect of ATP
on PFK 1 ?