LEGEND:
 = Clinical correlation;
#
  = Step number (Glycolysis)
 = Positive regulation
▬ = Negative regulation
↑ = increase/d
↓ =decrease/d
Glucose: C 6 H 12 O 6
• Beginning or end of major pathways of carbohydrate
metabolism
• Major form in which carbohydrates absorbed form GIT is
presented to cells
• Major fuel for the brain
• Biomedical importance
o Provide ATP in absence of oxygen allowing tissues
to survive anoxic episodes
o Heart muscle is adapted to aerobic performance
 ↓ glycolytic pathway; poor survival under
conditions of ischemia or myocardial
infarction
 hemolytic anemia: pyruvate kinase deficiency
 fatigue: phosphofructokinase deficiency
 cancer cachexia: ↑ lactate; ↑ gluconeogenesis;
hypermetabolism
 lactic acidosis: impaired activity of pyruvate
dehydrogenase
Relationship of Glucose to Major pathways of
Carbohydrate Metabolism
GLYCOLYSIS
 (Harper’s Illustrated Biochemistry Chapter 18)
• A.k.a. Embden-Meyerhoff Pathway
• A Catabolic Pathway (TCA = Amphibolic Pathway)
• Used by all cells to extract energy from glucose
• Aerobic or Anaerobic
A) Aerobic glycolysis –Pyruvate will be oxidized to CO2,
H2O, and Energy (ATP);
B) Anaerobic glycolysis – Pyruvate is converted to
Lactate utilizing NADH to NAD+ which is used by
Glyceraldehyde to form 1,3bis-Phosphoglycerate.
SUBJECT: BIOCHEMISTRY
TOPIC: CARBOHYDRATE
M ETABOLISM 1 (GLYCOLYSIS &
GLUCONEOGENESIS)
LECTURER: DR. UY
DATE: DECEMBER 2010
3 Stages of Glycolysis
I. Primary Stage
D-Glucose + 2ATP → D-fructose 1,6–Bisphosphate +
2ADP + 2H
• A.k.a. Trapping stage – trapping of glucose in form
of Glucose 6-Phosphate (G6P) with the utilization
of ATP. Phosphorylation with a negatively charged
PO4 to Glucose forming G6P, glucose is prevented
from moving outside of the cell therefore “trapping”
it inside the cell.
 In von Gierke disease, aka Type I Glycogensosis
(Glycogen storage disease) deficiency of Glucose-
6-Phosphatase (G6Pase) which catalyzes the
hydrolysis of G6P to D-Glucose, preventing G6P to
be converted to D-glucose which can be
transported outside the cell. This results in the
accumulation in the liver of excessive amounts of
normal glycogen.
II. Splitting Stage
D-fructose 1,6-Bisphosphate → 2 D-Glyceraldehyde 3-
Phosphate
• Splitting of FBP into two Triose phosphates
(G3P)
III. Oxido-reductase Phosphorylation Stage
2D-G3P + 4ADP + 2Pi + 2H + → 2Lactate + 4ATP +
2H 2 O
• Earning stage – formation of ATP
*Stage I and II are the investment stage (Investment
of 2 ATPs).
*In the energy generation phase 4 ATP and 2NADH will
produce 3ATP each = 6ATP in the ETC.
*Glycolysis yields a net of only 2 ATP because the other 2
 • GLUT4 – (in the muscles and adipose tissues)
Stage I and !! : insulin dependent transporters
Investment  In DM type 1 patients, lack of insulin causes
phase fatigue due to lack of ATP  caused by the
lack of glucose transported inside.
Stage III : Energy • GLUT2 – (in liver and pancreas)
generation phase

GLYCOLYSIS
STEP 1 - Phosphorylation by
Hexokinase/Glucokinase (Irreversible)
- Trapping of glucose by phosphorylation to G6P
DIFFERENCE BETWEEN HEXOKINASE AND
GLUCOKINASE
*This is where
the 1st ATP is invested.

GKRP attaches to glucokinase in its inactive form in the

nucleus – forming a complex with GK.
With the increase in glucose concentration in the cell,
1   release of GK by GKRP into the cytosol is promoted –
converting GK to its active form. While the increase of
Fructose 6-Phosphate (F6P) signals the inactivation of GK
back into the nucleus with GKRP therefore inhibiting its
activity.
 M ATURITY -O NSET D IABETES OF THE Y OUNG (MODY)
Type 2
An autosomal dominant disorder involving mutations in
the glucokinase (GCK) gene.

Hexokinase: Patients have progressive hyperglycemia that is usually

- Low Km for glucose relative to its concentration in asymptomatic at diagnosis and is usually managed
blood. (high affinity for glucose) with diet alone.
- Abundant in the Muscles STEP 2 – Isomerisation by Phosphoglucose
o Hexokinase immediately reacts even with a isomerise (Reversible)
low concentration of glucose.
o This rapid reaction of the enzyme is
important for G6P to be readily available in
the muscles.
- Strongly inhibited by its product Glucose 6-
Phoshate (G6P) hence its reaction is not at
equilibrium.
o When there is enough G6P in the muscle
Hexokinase is inhibited. (Low Vmax)
- In liver, hexokinase is saturated under normal
condition
Glucokinase (GK): 2
 
- S 0.5 for glucose is higher than the Km for glucose of
other kinases
- less sensitive to product inhibition by G6P
(G6P does not inhibit Glucokinase)
- contribute to the capacity of the liver to “buffer”
blood glucose levels because of the following
features
o When there is too much glucose it will be
released.
A. High (Km) S 0.5 for glucose
STEP 3 – 2 nd Phosphorylation by PFK-1 (Rate
Limiting Step)
3
  During starvation, The bifunctional enzyme is
• During this step the 2nd ATP is invested; And F6P is
phosphorylated with Phosphofructokinase-1 (PFK-
1) into Fructose 1,6-bisphosphate (FBP).
• Regulatory step / Rate limiting step / 1st committed
step of glycolysis.
▬ Abundance of ATP and Citrate regulates
glycolysis at this step by inhibiting the activity of
PFK-1.
 Increased amount of AMP occurs during starvation
which means there is a high demand of ATP
therefore promoting the activity of PFK-1.
 Fructose 2,6-bisphosphate (FBP) increases
during fed state which promotes PFK-1 activity.
(See Diagram below and explanation continued for FBP.)
o How Fructose 2,6-bisphosphate ↑PFK-1
is decreased  which causes a decrease in (↓)
cAMP and decreasing the activation of (↓) PKA
which causes increased (↑) dephosphorylation of
a bifunctional enzyme Phosphofructokinase-
2(PFK-2)/Fructose 1,6-Bisphosphatase-
2(FBP-2) complex (active PFK-2; inactive FBP-2).
PFK-2 when active converts F6P into Fructose 2,6-
Bisphosphate which increases activity of PFK-1. .
∴↑Glucose = ↑ Glycolytic activity
o On the opposite effect... (not shown in the diagram)
phosphorylated deactivating PFK-2 and activating
FBP-2 which converts Fructose 1,6-
bisphosphate to F6P (a reverse of the function of
PFK-1); decreasing the activity of PFK-1.∴↓Glucose =
↓ Glycolytic activity
 PHOSPHOFRUCTOKINASE (PFK-1) DEFICIENCY
- A form of glycogen storage disease or Glycogenosis
(Type VII)
- *Defined: Glycogenosis - Any of the (12 types of)
glycogen deposition diseases characterized by
accumulation of glycogen of normal or abnormal
chemical structure in tissue
- Normal glycogen accumulates in muscles
- Results in inefficient use of glucose stores by RBCs
and muscles
- Patients experience hemolytic anemia and muscle
cramping
*’bis-‘ indicates phosphate groups are separated, whereas
‘di-‘ indicates that the phosphate groups are joined e.i. ADP.
STEP 4 – Cleavage by Aldolase
A.k.a. the splitting stage as previously mentioned in page 1
 ALDOLASE A ( found in RBCs and muscle)
- Absence of the enzyme (aldolase) may cause
nonspherocytic hemolytic anemia.
*most/almost all of glycolytic enzyme deficiencies
manifest hemolytic anemia because this causes the
‘pumps’ in the membrane of RBC do not function
properly. This allows the RBC to be exposed to 7
 
oxidative properties which causes hemolysis or
swelling of the RBC.
*In this disease however the hemolytic anemia is
nonspherocytic which means the RBCs are not
spherical or sphere-shaped like most hemolytic
anemia.
 TRIOSE PHOSPHATE ISOMERASE (TPI)
- Patients with TPI deficiency have neonatal hemolytic
anemia and progressive neurologic involvement
- Progressive hypotonia with eventual diaphragm 8
 
paralysis and cardiomyopathy.
STEP 5 – Most DHAP are converted to G3P
which is utilized in the glycolytic pathway.

x  2  

9  

4  

5  

10  
*because most DHAP is converted to G3P there should be
2 molecules of G3P converted to Pyruvate.

STEP 6 – oxidation by G3P dehydrogenase

As previously mentioned most of the DHAP will be
converted to G3P, which G3P is the one utilized in the
formation of Pyruvate, therefore from everything beginning
in step 5 happens in pair.

In this step, G3P is converted to a high

6
  energy compound 1,3- x
 2
 
Bisphosphoglycerate (1,3BPG) by
Glyceraldehyde 3-phosphate dehydrogenase
(G3PDH or GAPDH) the reduction of NAD + to NADH+H +
which will form (3)ATP* in the ETC or utilized by the
reduction of pyruvate to lactate catalyzed by lactate
dehydrogenase in anaerobic glycolysis. *2(NADH+H+)
therefore, 6 ATPs
STEP 7
  –
  1 st substrate level Phosphorylation by During starvation, glucagon is increased. This stimulates
Phosphoglycerate kinase (PGK) (and ADP ATP) the activity of adenylyl cyclase which converts ATP to
and formation of 2,3BPG byproduct. cAMP. cAMP then activates PKA. PKA phosphorylates
Pyruvate kinase(PK) turning it into its inactive form.
At this step, Therefore, with decreased glucose glycolysis decrease
1,3BPG is converted to 3-Phosphoglycerate by production of pyruvate with the inactivation of PK.
transferring of the phosphate to ADP forming ATP. This is *kinases are inactive when phosphorylated similar to PFK-2
the first substrate level phosphorylation in Glycolysis. 2ATPs
should be formed because there are 2 molecules of During Anaerobic glycolysis pyruvate forms Lactate
1,3BPG from the splitting. At this point of glycolysis the catalyzed by the enzyme Lactate dehydrogenase
2ATPs invested in steps 1 and 3 has already been regained. utilizing with it. NADH+H+ from the activity of G3P
dehydrogenase
There is also the presence of an enzyme mutase,
abundant in the RBC, which may form a byproduct 2,3BPG
from 1,3BPG. 2,3BPG shifts the oxygen dissociation curve
to the right – releasing more oxygen to the tissues. 2,3BPG
can then be converted to 3-Phosphoglycerate(3PG)
with the use of the enzyme Phosphatase.
*2,3BPG or diphosphyglycerate/DPG is also likely an
intermediate of the reaction.
*However, this pathway does not produce ATP.
*PGK is inhibited by arsenate
 Arsenic toxicity – arsenate competes with the
inorganic phosphate (Pi) hydrolyzing 3PG to 2PG
without ATP formation
STEP 8 – Isomerization by Phosphoglycerate 2 ATP
mutase consumption
with the
3-Phosphoglycerate is isomerized to 2-Phosphoglycerate
enzymes
STEP 9 – Dehydration catalyzed by Enolase Hexokinase/Glu
cokinase and
2-Phosphoglycerate removes H2O to form a high energy
Phosphofructoki
phosphate compound  phosphoenol pyruvate(PEP)
nase-1(PFK-1).
*enol compounds=high energy compounds
 Enolase is inhibited by Flouride; used to inhibit
2NADH+2H +
glycolysis in blood samples used for measuring glucose.
produced from
STEP 10 – 2 nd Substrate level phosphorylation G3P
(Irreversible) dehydrogenase.
At this step, phosphate is transferred from PEP to ADP by
pyruvate kinase(PK) – forming 2 molecules of ATP per
glucose. Since the invested ATPs were already regained,
this 2nd substrate level phosphorylation is the first gaining
of ATP from glucose. PK is allosterically activated by
FBP(product of the rate limiting step – page2)
 Pyruvate Kinase Deficiency & Hemolytic
Anemia
Lack of ATP affects ion pumps especially Na+/K+
ATPase. –causing the cells to swell & lyse 4ATP
produced
from
(2)Phospho-
glycerate
kinase (PGK)
and (2) (PK
Pyruvate
kinase)
In anaerobic
pathway, 2
NADH from
G3PDH is
utilized to
convert
3 I RREVERSIBLE STEPS OF GLYCOLYSIS WHICH ARE ALSO
REGULATORY STEPS
*All are kinase enzymes.
NADH Generated by Glycolysis has to be oxidized back to
NAD+. This is done by the enzyme Lactate
Dehydrogenase(anaerobic) & Substrate Shuttles(aerobic).
In Aerobic glycolysis, NADH produced by G3PDH needs to
be transported into the mitochondria for ATP synthesis in
the ETC, however, NADH is NOT permeable to the inner
mitochondrial membrane. By utilizing substrates (Malate
and Aspartate) permeable to the inner mitochondrial
membrane, NADH is made available to the ETC in the
mitochondria.
OAA + NADH →Malate Dehydrogenase→ Malate + NAD+
NADH is oxidized back to NAD+ with Malate
dehydrogenase (MDH) reducing Oxaloacetate OAA to
Malate which is permeable to the inner mitochondrial
which is also permeable to the inner mitochondrial
membrane then exits the mitochondria and converted back
to OAA by AST.
Fate of the PYRUVATE
Carboxylation of Pyruvate to
Oxaloacetate (OAA) is
catalyzed by Pyruvate
carboxylase.
(Oxidative) Decarboxylation of Pyruvate to Acetyl CoA is
catalyzed be Pyruvate deydrogenase complex along with
the reduction of NAD+. Acetyl CoA will proceed to TCA cycle.
The Pyruvate dehydrogenase complex is a multienzyme
complex which consists of Pyruvate Dehydrogenase,
Dihydrolypoyl Tranacetylase, Dihydrolipoyl Dehydrogenase.
(The succeeding discussion on Oxidative decarboxylation was not
discussed but it was in the PPT and is also found in the Harpers
on chapter 18, page 153, “The oxidation of pyruvate to Acetyl-CoA
is the irreversible route from glycolysis to the citric acid cycle”.)
During the conversion the intermediates do not dissociate
and remain bound to the components of the multienzyme
complex.
MECHANISM: (OXIDATIVE CARBOXYLATION OF PYRUVATE)
Pyruvate is decarboxylated to a hydroxyethyl (Acyl) bound to
thiamine diphosphate (TPP or TDP) with the enzryme
Pyruvate dehydrogenase, forming Acyl-TPP. Acyl-TPP reacts
with an oxidized lipoamide (Lip-S2) catalyzed be the
Dihydrolipoyl Transacetylase component, forming Acyl-
Lipoate/Acetyl lipoamide (in Harper’s). Acyl-lipoate will then
reacts with Coenzyme A(CoA-SH) forming Acetyl CoA and
the reduced lipoamide. However lipoamide has to be
returned to its original form which is reoxidized by the
Dihydrolipoyl Dehydrogenase(flavoprotein component
containing an FAD, forming FADH and Lip-S2. Dihydrolipoyl
Dehydrogenase is finally oxidized by NAD+ for it to return to
its orginal form – flavoprotein containing FAD.
 ATP per
Pathway
Reaction Catalyzed Method of ATP
Mol of GLUCONEOGENESIS (Harper’s Illustrated Biochemistry Chapter
by Formation 20)
Glucose

Glyceraldehyde 3-
Respiratory chain
• Defined: The net synthesis or formation of glucose
Glycolysis phosphate
oxidation of 2 NADH
6* from non-carbohydrate substrates:
dehydrogenase
- Amino acids (Except lysine & leucine)
Phosphoglycerate kinase
Substrate level
2 - Lactate
phosphorylation
- Pyruvate
Pyruvate kinase
Substrate level
2 - Propionate
phosphorylation
- Glycerol
10 - Fructose
Consumption of ATP for reactions of hexokinase and • Essential for survival of humans and other animals
–2
phosphofructokinase

Net 8* Enzymes Unique To Gluconeogenesis (Irreversible

Citric acid
Pyruvate dehydrogenase
Respiratory chain
6*
reactions)
cycle oxidation of 2 NADH
(The rest may be found in Glycolysis)
Isocitrate Respiratory chain
6*
dehydrogenase oxidation of 2 NADH • Mitochondrial:
-Ketoglutarate Respiratory chain
6* 1. Pyruvate Carboxylase – cannot act in the cytosol
dehydrogenase oxidation of 2 NADH
 Pyruvate→OAA
Substrate level
Succinate thiokinase 2
phosphorylation • Cytosolic:
Succinate Respiratory chain
4 2. PEP Carboxykinase (PEPCK)
dehydrogenase oxidation of 2 FADH2
 OAA →PEP
Respiratory chain
Malate dehydrogenase 6
oxidation of 2 NADH 3. Fructose-1,6-Bisphosphatase (FBPase)
Net 30  FBP → F6P
Total per mol of glucose under aerobic conditions 38
4. Glucose-6-Phosphatase (G6Pase)
Total per mol of glucose under anaerobic conditions 2
 G6P → Glucose
(Values are changed assuming 2ATPs from FADH and 3 from NADH)
You may refer to Harper’s Ill. Biochem: Figure 20-1 for a more
6-Phosphofructo-1-kinase is a Regulatory Enzyme detailed figure.
of Glycolysis
▬ Negative Allosteric Effectors
1. Citrate
2. ATP
3. Hydrogen ions (low pH)
Positive Allosteric Effectors
1. AMP
2. Fructose 2,6 – bisphosphate

Rate of Glycolysis Depends On:

Effect on Rate
↑ATP = ▬
Energy state of the cell (ATP or AMP)
↑AMP = 
Internal environment of the cell (H
↑H+= ▬
ions) ↑H+=acidosis
Availability of alternate fuels such as
↑Citrate = ▬
fatty acids and ketone bodies (citrate)
Insulin / glucagon ratio in the blood ↑Insulin/Glucagon
(F - 2,6 – BP) Ratio = 
(For the Glycolysis summary please refer to slide 49 of the PPT)
Remember:
-Aerobic
-Anaerobic Lactate as substrate for gluconeogenesis
leading to
Lactic
Acidosis
-Regulatory
steps
-Mutations
 2Lactate + 6ATP + 6H2O → Glucose + 6ADP + 6Pi + 4H+
Glucose can be immediately trapped by the muscle with the
enzyme Hexokinase. During exercise there will be an
increased amount of lactate causing lactic acidosis. This
will cause pain in the muscles (commonly in the form of
cramps). Because of muscles do not have lactate
dehydrogenase Lactate will be transported to the liver
where it can be converted to glucose. In the liver lactate
dehydrogenase can catalyze the oxidation of Lactate to
Pyruvate which requires 6ATP to form glucose
(gluconeogenesis) to be sent back to the muscle and other
tissues. Utilizing 6ATP makes gluconeogenesis an ‘expensive’
pathway.
Amino acids as substrate for Gluconeogenesis
Carboxylation of Pyruvate is catalyzed by Pyruvate
carboxylase by transferring the CO2 attached to biotin
forming Oxaloacetate (OAA).
Because OAA is not permeable to the mitochondrial
membrane it is reduced by NADH to malate.
In the cytosol, malate is reoxidized to OAA with NAD+.
OAA will then be converted (decarboxylation and
phosphorylation) to Phosphoenol pyruvate (PEP) by
PEP carboxykinase (PEPCK) with the cofactor GTP(→GDP).
The succeeding steps beginning from PEP are reverse
reactions of glycolysis,
except in FBPase &
G6Pase.

In the muscles Alanine transaminase or Alanine ATP is again utilized

aminotransferase (ALT) catalyze the transferring of an in the formation of
amino group from glutamate to pyruvate forming α- 1,3-BPG.
ketoglutarate and alanine. Through the blood Alanine Because 2 molecules
travels to the liver where it is converted back to pyruvate is required in the
with ALT. Aside from the 6 ATP required for pyruvate to formation of FBP 2
undergo gluconeogenesis, Ammonium needs to be pyruvate must be
released from glutamate through urea cycle utilizing 4 ATP. utilized to form 1
This makes amino acids as substrate more expensive (ATP). molecule of glucose.
2Ala + 10ATP + CO2 → Glucose + Urea + 10ADP + 10Pi After this step, 4 ATP
and 2GTP has been
utilized by the pathway.
2NADH will be
utilized to convert 2
molecules
of1,3BPG to form 2
molecules of G3P.
PFK-1 is an
irreversible enzyme
of glycolysis
therefore
gluconeogenesis
needs its own
enzyme FBPase.
 Excessive amounts of ammonia loaded into the liver, up
to a point that not everything can be immediately
converted to urea can cause comatose due to hepatic The last step of gluconeogenesis is catalyzed by Glucose-
encephalopathy. This is common in liver cirrhosis 6-phosphatase(G6Pase) which dephosphorylates G6P
caused by alcoholism. to Glucose. G6P is not found in the muscles therefore,
GLUCOKINASE IN THE LIVER gluconeogenesis cannot occur in the muscle
• Regulates blood glucose after a meal  von Gierke’s Disease (mentioned in page 1)
- type I glycogen storage disease(GSD)
• Promotes increased hepatic utilization of glucose - deficiency of glucose-6-phosphatase
o Promotes Glycolysis not gluconeogenesis - severe hypoglycemia causing lethargy,seizures and
nor glycogenolysis. brain damage
- hepatomegaly, increased bleeding (clotting factors
2,6,7,9,10 and platelets are affected) and growth
retardation
Glucagon  Main
gluconeogenic
hormone

Similar to the
previous discussion
in page 3 and 5,
attachment of the
hormone glucagon
deactivates PK
↑Catecholamine increase secretion of Glucagon which is
synthesized from α-pancreatic cells.
During starvation a great amount of ATP is used for
gluconeogenesis which may be taken from the oxidation of
fatty acids in the liver. This process however forms ketone
bodies.
Insulin

• the only counter-regulatory hormone in gluconeogenesis

• main glycolytic hormone
G LUCOSE IS S YNTHESIZED FROM M OST A MINO A CIDS
• action
• All amino acids except leucine & lysine can supply o binds to receptor
carbon for the net synthesis of glucose by o receptor sends signal activating phosphoprotein
gluconeogenesis phosphatase
• Pyruvate, oxaloacetate – catabolic products of amino o inactive PFK-2 becomes active PFK-2 (Pi removed)
acids from which glucose synthesis can occur o active PFK-2 increases concentration of F-2,6 BP
o F-2,6 BP binds to PFK-1 & increases its activity
• Anaplerotic Reactions – those that lead to net o Rate of glycolysis is increased
synthesis of TCA intermediates
Alcohol Oxidation Inhibits Gluconeogenesis
 Hepatic encephalopathy – utilization of amino
acids for gluconeogenesis may cause production of Due to excess NADH forcing the equilibrium of the
excess urea which may lead to an encephalopathy reactions catalyzed by lactate dehydrogenase and malate
associated with cirrhosis of the liver, attributed to the dehydrogenase
passage of toxic nitrogenous substances from the →
portal to the systemic circulation; cerebral
manifestations may include coma.
 PEPCK Deficiency
Gluconeogenic Ketogenic Both
- rare but severe metabolic defect
Glycine Leucine Threonine
Serine Lysine Isoleucine - absence of the cytosolic form results in cerebral
Valine Phenylalanine atrophy, optic atrophy, fatty infiltration of the liver
Histidine Tyrosine and kidney and intractable hypoglycemia (including
Arginine Tryptophan lost of vision)
Cysteine  Fructose-1,6-Bisphosphatase Deficiency
Proline
Hydrodyproline - Presents with neonatal hypoglycemia, along with
Alanine acidosis, irritability, tachycardia, dyspnea, hypotonia,
Glutamate moderate hepatomegaly.
Glutamine - typically only the liver enzyme is deficient, the muscle
Aspartate activity is normal
Asparagines
(Please refer to slide 82 of the PPT for the gluconeogenesis
Methionine
summary)↓

Glucose can be synthesized from Propionyl-CoA

(odd-numbered carbon)
• Propionyl-CoA – good precursor for gluconeogenesis as
it yields oxaloacetate by anaplerotic pathway(TCA cycle).
• Triacylglycerol(TAG) – when hydrolyzed yields 3 FA’s &
glycerol (a substrate for gluconeogenesis)
o Because Glycerol kinase(found in adipose tissue)
is absent in the liver, TAG needs to be hydrolyzed
to FAs and glycerol before becoming a substrate
for gluconeogenesis.