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CARBOHYDRATES:
GLYCOLYSIS
OVERVIEW
A. Glycolysis
- function as the initial step in the metabolism of glucose to produce energy for the cell
- process in which glucose molecules  2 pyruvate molecules
- results in the net ATP production (also consumed at several stages)
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B. Pyruvate
- end product of glycolysis in cells with mitochondria and an adequate supply of oxygen
C. Aerobic Glycolysis
- oxygen is required to reoxidize the NADH formed during the oxidation of glyceraldehyde 3-phosphate
- sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA
- produces 6 moles ATP / mole of glucose
D. Anaerobic Glycolysis
- glucose  pyruvate  reduced by NADH  lactate
- no net formation of NADH therefore it can occur without oxygen
- allows the continued production of ATP (2 moles ATP/mole of glucose) in tissues that lack mitochondria
(RBCs) or in cells deprived of sufficient oxygen

E. Stages of Glycolysis

1. Conversion of Hexose to Triose Phosphate

- 6 carbon sugar is split to 2 three-carbon sugar
- requires 2 moles ATP/mole of hexose that is split
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2. Conversion of Triose Phosphate to Pyruvate
- 2 three-carbon sugars metabolized to 2 molecules of pyruvate
- produces 2 moles ATP/mole of triose phosphate converted to pyruvate ( 4 moles ATP
produced/mole of hexose)

SOURCES of CARBON and ENERGY for GLYCOLYSIS

A. Carbon Sources
1. Starch
- major nutrient derived from ingested plant cells
2. Lactose
- disaccharide of glucose and galactose
- major carbohydrate component of milk
3. Fructose
- ketose isomer of glucose
- from fruits
- important carbohydrate source when sucrose (glucose-fructose disaccharide) intake is high
B. Glucose
- product of starch digestion
- major form in which carbohydrate is presented to the cells of the body

C. Glycogen
- major form of carbohydrate storage in animals
- highly branched polymer of glucose

REACTIONS of GLYCOLYSIS
- 2 molecules of NADH are formed when pyruvate is produced
- NADH is reconverted to NAD+ when lactate is the end product

A. Phosphorylation of Glucose
- irreversible reaction effectively trapping glucose as glucose 6-phosphate (too polar  does not diffuse
out of the cell)
- lack of specific carriers  phosphorylated sugar molecules do not readily penetrate cell membranes
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1. Hexokinase I
- ubiquitous
- catalyzes phosphorylation of glucose in most tissues (transfers a phosphoryl group from ATP to
the C6-OH group of glucose)
- 1 of 3 regulatory enzymes of glycolysis
a. Substrate Specificity and Product Inhibition of Hexokinase
- phosphorylate several hexoses other than glucose
- feedback inhibited by glucose 6-phosphate
b. Kinetic Properties of Hexokinase
- low Km (easily saturable)  high affinity to glucose  efficient phosphorylation and
subsequent glucose metabolism even when tissue concentration of glucose is
low
- low Vmax for glucose  cannot phosphorylate large quantities of glucose
2. Glucokinase (Hexokinase D or Type IV)
- in - liver parenchymal cells
- cells of pancreas
- predominant enzyme for glucose phosphorylation (very specific for glucose)
- functions as glucose sensor in the cells of pancreas  determine threshold for insulin
secretion
- levels increased by
- insulin
- carbohydrate-rich diet
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a. Kinetics
- higher Km (not easily saturable)  requires higher [glucose] for half-saturation 
functions only with elevated hepatocyte [glucose]
- functions only with elevated intrahepatocyte glucose concentration
- high Vmax  allows liver to effectively remove glucose from the portal blood 
minimizes hyperglycemia during absorptive period

i. GLUT-2
- insures that blood glucose equilibrates rapidly across the hepatocyte membrane
b. Regulation by Fructose 6-Phosphate and Glucose
- not allosterically inhibited by glucose 6-phosphate
- indirectly inhibited by fructose 6-phosphate (in equilibrium with glucose 6-phosphate)
- stimulated indirectly by glucose (carbohydrate-rich diet)
i. Glucokinase Regulatory Protein
- in hepatocyte nucleus
- presence of fructose 6-phosphate  glucokinase translocation into nucleus 
tightly binds with the regulatory protein  enzyme inactivation
- increased blood glucose concentration  GLUT-2 transport  increased
intrahepatocyte glucose concentration  glucokinase release from
regulatory protein  glucokinase in the cytosol  glucose
phosphorylation into glucose 6-phosphate
- fall of glucose concentration and presence of fructose 6-phosphate 
glucokinase translocation into nucleus  tightly binds with the
regulatory protein  enzyme inactivation
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c. Regulation by Insulin
- meal  increased glucose concentration  cells of pancreas stimulated  insulin
release into the portal circulation
- ½ of newly secreted insulin extracted by the liver during the first pass to the
liver
i. Insulin
 glucokinase gene transcription  increased liver enzyme protein 
increased enzymatic activity
ii. Diabetes Mellitus
 hepatic glucokinase deficiency  inefficiency to decrease blood glucose
levels
3. Glucose 6-Phosphate
- intermediate of pivotal importance
- precursor for several other metabolic pathways both anabolic and catabolic
B. Glucose 6-Phosphate Isomerization
- catalyzed by phosphoglucose isomerase
- readily reversible
- controlled by substrate-product levels (stoichiometric control)
- proceeds via an enediolate intermediate
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C. Fructose 6-Phosphate Phosphorylation
- irreversible reaction
- catalyzed by phosphofructokinase 1 (PFK-1) which is the major regulatory enzyme of glycolysis
- most important control point of glycolysis
- rate-limiting step of glycolysis
- controlled by
- ATP
- fructose 6-phosphate
- regulatory substances (effectors or modulators)

1. Regulation of PFK-1 by Energy Levels Within the Cell

a. Inhibited By
i. Increased ATP
- decreases affinity of PFK-1 for fructose 6-phosphate
- signals presence of high energy
ii. Elevated Citrate Levels
- high ATP levels  citrate not metabolized  enters cytoplasm  signal high
energy state
b. Activated By
- increased AMP (signals low energy state)
- fructose 6-phosphate
- fructose 2,6-biphosphate
2. Regulation of PFK-1 by Fructose 2,6-Biphosphate
i. Fructose 2,6-Biphosphate
- formed from fructose 6-phosphate when high levels are present
- most potent activator of PFK-1 (in the liver only)
- increases the affinity of PFK-1 to fructose 6-phosphate
- diminishes the inhibitory effect of ATP
- inhibitor of fructose 1,6-biphosphatase
- formed by PFK-2
- converted back to fructose 6-phosphate by fructose biphosphatase-2
ii. PFK-2
- inhibited by
- citrate
- ATP
- phosphorylation by cAMP-dependent protein kinase
a. During Well-Fed State
- carbohydrate-rich meal  decreased glucagon levels, increased insulin levels 
increased fructose 2,6-biphosphate, increased rate of glycolysis
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b. During Starvation
- elevated glucagon level, decreased insulin level  decreased intracellular hepatic
fructose 2,6-biphosphate  decreased glycolysis, increased gluconeogenesis

D. Fructose 1,6-Biphosphate Cleavage

- to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate
- catalyzed by aldolase A
- unregulated reversible reaction
- aldolase operates via Schiff base and enamine intermediates in animals and plants
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1. Aldolase B
- in the - liver
- kidney
- cleaves fructose 1,6-biphosphate
- functions in the metabolism of dietary fructose
E. DHAP Isomerization
- interconversion of DHAP and glyceraldehyde 3-phosphate  net production of 2 molecules of
glyceraldehyde 3-phosphate
- catalyzed by triose phosphate isomerase through an enediolate intermediate
- glyceraldehyde 3-phosphate is further metabolized in the glycolytic sequence
- reversible

F. Glyceraldehyde 3-Phosphate Oxidation

- to 1,3-biphosphoglycerate
- catalyzed by glyceraldehyde 3-phosphate dehydrogenase
- reversible
- 1st redox reaction of glycolysis
- require NAD+ as electron carrier
- limited amount of NAD+ in the cell  NADH must be reoxidized to NAD+ for glycolysis to continue
a. 2 Major Mechanisms of NADH Oxidation
i. NADH-linked conversion of pyruvate to lactate
ii. Oxidation via respiratory chain
1. Substrate Level Phosphorylation
- oxidation of aldehyde group of glyceraldehyde 3-phosphate to a carboxyl group is coupled to
the attachment of Pi to the carboxyl group  high-energy phosphate production
(coupled directly to the oxidation reaction)  drives the production of ATP in the next
reaction of glycolysis
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2. Arsenic
- toxicity by enzyme inhibition (pyruvate dehydrogenase which requires lipoic acid as cofactor)
a. Arsenate
- pentavalent arsenic
- can uncouple oxidation and phosphorylation at this step
- can substitute for Pi in the phosphorylation reaction by combining with an energy-rich
thioester intermediate to form an unstable compound (1-arseno-3-
phosphoglycerate)  3-phosphoglycerate  synthesis and dephosphorylation
of 1,3-biphosphoglycerate bypassed  energy deprivation obtained from this
pathway
3. 2,3-Biphosphoglycerate Production
- 1,3-biphosphoglycerate  2,3-biphosphoglycerate
- catalyzed by biphosphoglycerate mutase
- trace amounts in most cells
- high amount in RBCs
- hydrolyzed by a phosphatase  3-phosphoglycerate (intermediate of glycolysis)
- shunt reaction included in RBC glycolysis
G. ATP Formation from 1,3-Biphosphoglycerate (1,3-BPG) and ADP
a. 1,3-Bisphosphoglycerate
- “high-energy” mixed anhydride of a phosphate and a carboxylic acid
- catalyzed by phosphoglycerate kinase
- reversible reaction
- converts 1,3-BPG  3-phosphoglycerate (3-PG)
- 1st glycolytic step that generates ATP
- substrate level oxidative phosphorylation
- 2 molecules of 1,3-BPG formed from each glucose molecule  2 ATP molecules produced  replaces
the ATP molecules consumed in the earlier formation of glucose 6-phosphate and fructose 1,6-
biphosphate
H. Phosphate Group Shift from Carbon 3 to Carbon 2
- catalyzed by phosphoglycerate mutase (requires cofactor - trace amounts of 2-3, BPG)
- freely reversible
I. 2-Phosphoglycerate Dehydration
- catalyzed by enolase
- 2-phosphoglycerate  phosphoenolpyruvate (PEP) (contains high-energy enol phosphate)
- reversible reaction
J. Pyruvate Formation
- catalyzed by pyruvate kinase
- its equilibrium favors the formation of ATP
- liver isoenzyme is inducible (high carbohydrate intake, high insulin  increased enzyme
concentration)
- 3rd irreversible reaction of glycolysis
- substrate level phosphorylation
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1. Feed-Forward Regulation
- increased PFK activity  increased fructose 1,6-biphosphate levels  pyruvate kinase
activation

2. Covalent Modulation of Pyruvate Kinase

- low blood glucose levels  glucagon elevation  increased intracellular cAMP level  cAMP-
dependent protein kinase  pyruvate kinase phosphorylation  inactivation
(inhibition of hepatic glycolysis by glucagon)  PEP unable to continue in glycolysis
 enters gluconeogenesis
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3. Phosphoprotein Phosphatase  dephosphorylated pyruvate kinase  enzyme reactivation
4. Inhibitors
- ATP
- fatty acids
- alanine
- acetyl CoA
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ALTERNATE FATES of PYRUVATE
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A. Oxidative Decarboxylation of Pyruvate
- catalyzed by pyruvate dehydrogenase complex
- important pathway in tissues with high oxidative capacity (cardiac muscle)
- irreversibly converts pyruvate to acetyl CoA
- acetyl CoA
- major fuel for the TCA cycle
- building block for fatty acid synthesis
B. Carboxylation of Pyruvate to Oxaloacetate (OAA)
- catalyzed by pyruvate carboxylase (biotin dependent)
- replenishes TCA cycle intermediate
- provides substrates for gluconeogenesis
C. Reduction of Pyruvate to Ethanol (Yeast, Certain Microorganisms)
1. 2 Step Process
a. Pyruvate Decarboxylase Reaction
- pyruvate is decarboxylated to acetaldehyde and carbon dioxide
- with the aid of the coenzyme thiamine pyrophosphate, which stabilizes the reaction’s
carbanion intermediate
b. Alcohol Dehydrogenase Reaction
- acetaldehyde is reduced with NADH to form ethanol and NAD +
- released CO2 leavens (raises) bread
- ethanol is used to make alcoholic beverages
D. Pyruvate to Alanine
- links carbohydrate and amino acid metabolism
- catalyzed by alanine aminotransferase

E. Reduction of Pyruvate to Lactate

1. Lactate
- final product of anaerobic glycolysis
- catalyzed by LDH
- major fate for pyruvate in
- RBCs
- testes
- WBCs
- lens and cornea of the eye
- renal medulla
2. Muscle Lactate Formation
- exercising muscle  NADH production (by glyceraldehyde 3-phosphate DH and the 3 NAD+-
linked dehydrogenases of the TCA cycle) exceeds the oxidative capacity of the
respiratory chain  elevated NADH/NAD+ ratio  reduction of pyruvate to lactate
favored  lactate accumulation  decreased pH  muscle cramps and pain (much of
lactate diffuses into the blood stream)
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3. Lactate Consumption
- direction of LDH reaction depends on
- relative intracellular concentrations of pyruvate and lactate
- NADH/NAD+ ratio
- liver, heart  lower NADH/NAD+ ratio (than exercising muscle)  oxidation  pyruvate
- liver  pyruvate  glucose (by gluconeogenesis) or oxidized in the TCA cycle
- heart muscle  lactate  CO2 + H2O via TCA cycle

ENERGY YIELD of GLYCOLYSIS

A. Anaerobic Glycolysis
- anaerobic catabolism of glucose can be 100 times faster than the catabolism of glucose in the presence of
oxygen

Glucose + 2Pi + 2ADP  2Lactate + 2ATP + 2H2O

1. ATP Consumed
- 2 moles ATP/mole of glucose used to form fructose 1,6-biphosphate
2. ATP Production
- 2 moles ATP/mole of glucose produced by phosphoglycerate kinase reaction
- 2 moles ATP/mole of glucose produced by pyruvate kinase reaction
 net production of 2 moles ATP
- valuable source of energy under several conditions
a. Limited Oxygen Supply (intensive exercise)
b. Tissue with Few or No Mitochondria
- renal medulla
- mature RBCs
- WBCs
- cells of the
- lens
- cornea
- testes
 release only small fraction of energy (2 ATP molecules/glucose molecule converted to lactate)
3. NADH Production
- NADH produced by glyceraldehyde dehydrogenase  used up by LDH to reduce pyruvate to
lactate (2 lactate molecules produced per glucose molecule metabolized)  no net
NADH production
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B. Aerobic Glycolysis

Glucose + 2Pi + 2NAD+ + 2ADP  2 Pyruvate + 2ATP + 2NADH + 2H+ + H2O

1. ATP Consumed
- 2 moles ATP/mole of glucose used to form fructose 1,6-biphosphate
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2. ATP Production
- 2 moles ATP/mole of glucose produced by phosphoglycerate kinase reaction
- 2 moles ATP/mole of glucose produced by pyruvate kinase reaction
 net production of 2 moles ATP
- 3 or 5 moles ATP/mole of glucose produced by the NADH that is formed in the glyceraldehyde
3-
phosphate reaction
a. Glycerol 3-Phosphate Shuttle
- production of 1.5 moles ATP/mole of NADH
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b. Malate-Aspartate Shuttle
- production of 2.5 moles ATP/mole of NADH

3. Total Moles ATP Produced

- 5 or 7 moles
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ATP Formation in the Catabolism of Glucose
Pathway Reaction Catalyzed by Method of ATP ATP per Mol of Glucose
Formation
Glycolysis Glyceraldehyde Respiratory chain 3 or 5*
3-phosphate oxidation of 2 NADH
dehydrogenase
Phosphoglycerate kinase Substrate level 2
Pyruvate kinase phosphorylation 2
Total 7 or 9
Consumption of ATP for reactions of hexokinase and -2
phosphofructokinase
Net 5 or 7
*This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle. If the
glycerophosphate shuttle is used, then only 1.5 ATP will be formed per mol of NADH.

GLYCOLYSIS and RBC METABOLISM

A. Energy Metabolism
1. Mature RBCs contain no mitochondria  totally dependent on glycolysis for ATP production
2. ATP  support Na+ and K+-stimulated ATPase ion transport system  maintain proper RBC biconcave
shape
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B. Inherited Enzyme Deficiencies of Glycolysis
1. Pyruvate Kinase Deficiency
a. Normal Mature Erythrocyte
- lacks mitochondria
- completely dependent on glycolysis for production of ATP
b. ATP - high-energy compound
- required to meet the metabolic needs of the red blood cell
- fuel the pumps necessary for the maintenance of the biconcave and flexible shape of
the cell  squeeze through narrow capillaries
- 2nd most common (95%) cause (after glucose 6-phosphate dehydrogenase deficiency) of enzyme
deficiency-related hemolytic anemia
- restricted to the erythrocytes
- produces mild to severe chronic hemolytic anemia
- severe form requires regular red cell transfusions
- severity depends on
- degree of enzyme deficiency (generally 5-25% of normal levels)
- extent to which the RBCs compensate by synthesizing increased 2,3-BPG
levels
- almost all cases due to mutant enzyme with abnormal properties (altered kinetics)
c. Anemia in Glycolytic Enzyme Deficiencies
- due to reduced rate of glycolysis  decreased ATP production  altered red blood
cell membrane  changes in the shape of the cell  phagocytosis by the cells
of the reticuloendothelial system (particularly macrophages of the spleen)
- premature death and lysis of red blood cells  hemolytic anemia
2. Glucose Phosphate Isomerase Deficiency
- 4%
3. Triose Phosphate Isomerase Deficiency
- RBCs
- muscle cells
- WBCs
- CNS
4. Other Deficiencies
a. RBCs - pyruvate kinase deficiency
- hexokinase deficiency
- most patients with glycolytic enzyme deficiency  hemolytic anemia (varies depending on the specific
enzyme deficiency)  may not require therapy except for folic acid supplementation for severe
hemolysis
i. Pyruvate Kinase (PK) Deficiency - Case: Pyruvate Kinase Deficiency
Presentation
A newborn male infant is found to be anemic and mildly jaundiced
Blood Analysis
- some variability in RBC morphology
- below normal hemoglobin levels
- above normal reticulocyte count
Hemolysate Analysis
- pyruvate kinase activity is 20% of the normal level
Infant’s condition improved and was discharged. The infant is to be observed for
any increased severity of anemia. Splenectomy is to be considered if
severity of anemia increases
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Diagnosis
- genetic defect in pyruvate kinase activity
Discussion
- genetic deficiency of PK in RBCs (lack mitochondria  energy supply
depends on glycolysis)  mutant enzyme  abnormal properties
(most often altered kinetics, with only 5-25% PK activity) 
severely decreased glycolytic rate  low ATP production  low
energy to maintain RBC membrane structural integrity  shape
changes  phagocytosis by cells of the reticuloendothelial system
(particularly splenic macrophages)  premature RBC death and lysis
 hemolytic anemia
- reticulocytes
- immature RBCs
- retain their mitochondria
Prognosis
- no cure for the disorder but often survive to adulthood unless complications
exacerbate the condition
- severe cases
- blood transfusion and splenectomy may be necessary
ii. Hexokinase Deficiency
- ultimately reduces the amount of oxygen that is available for the tissues
- genetic defect of RBC hexokinase isoenzyme  low concentrations of glycolytic
intermediates (including 1,3-BPG, the precursor of 2,3-BPG)
- results to hemolytic anemia

LACTIC ACIDOSIS
A. Anaerobic Glycolysis as an Emergency Source of ATP
- circulatory collapse (myocardial infarction, pulmonary embolism, uncontrolled hemorrhage, shock) 
lack of oxygen  impaired oxidative phosphorylation (decreased ATP synthesis)  anaerobic
glycolysis (small amount of ATP production) which is life-saving during the period required to
re-establish blood flow  lactic acidosis
B. Blood Lactate
1. Normal Levels
- 1.2 Mm

2. Lactic Acidosis
- blood lactate level of 5 mM or more
- due to increased formation or reduced utilization  lowered blood pH  decreased bicarbonate
levels
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3. Oxygen Debt
- excess oxygen required in order to recover from a period when oxygen availability has been
inadequate
- often related to patient morbidity or mortality
- early detection by determining blood lactate levels
- blood lactate level determination
- determine presence and severity of shock
- monitor patient’s recovery
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HORMONAL REGULATION of GLYCOLYSIS
1. Short-Term Regulation
- allosteric activation or inhibition
- phosphorylation or dephosphorylation of rate-limiting enzymes
- minutes to hours effect
2. Hormonal Influences
- slower
- more profound effects
- 10-20 fold increase in enzyme activity
- occur over hours to days
A. Glycolytic Enzymes
- carbohydrate-rich meal consumption, insulin administration  increased gene transcription  increased
protein (enzyme) synthesis  increased amount and activity of liver glucokinase, PFK, PK 
favors glucose conversion to pyruvate (characteristic of well-fed state)
- increased glucagon, low insulin  decreased gene transcription and synthesis of glucokinase, PFK, PK
B. Gluconeogenic Enzymes
- glucagon (starvation, diabetes mellitus)  increased gene transcription  increased activity of PEP
carboxykinase, fructose 6-phosphatase, glucose 6-phosphatase
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BIOSYNTHETIC FUNCTIONS of GLYCOLYSIS
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SUMMARY