Carbohydrate Metabolism

Glycolysis, Gluconeogenesis
Pentose phosphate Pathway
Citric Acid Cycle
and
Oxidative Phosphorylation

Department of Biochemistry
 Carbohydrate metabolism

-Glycolysis (for catabolism)

Oxidation of glucose into pyruvate

-Gluconeogenesis (for anabolism)

Processes in glucose synthesis from nonhexose precursors

-Pentose phosphate pathway (for catabolism and anabolism)

Oxidative pathway of glucose for synthesis of specialized
products needed by the cell, NADPH and ribose 5-phosphate
for nucleic acids
 Glycolysis

Glycolysis : Greek, glykys = sweet, lysis = splitting

-A series of enzyme-catalyzed oxidation of glucose (C6)

yield two molecules of pyruvate (C3).

-During the sequential reactions of glycolysis, the free

energy is released from glucose oxidation, and conserved
in the from of ATP and NADH.

-Glycolysis is an universal pathway in glucose catabolism

found in most cells to obtain energy.
Glycolysis
Glycolysis
The reasons why intermediates in glycolysis
are phosphorylated.

4) pKa of phosphate group is low, giving each glyclytic

intermediates a net negative charge, and can not
diffuse out from the cell.

7) Phosphoryl groups are essential components in the

enzymatic conservation of metabolic energy.

10) Binding of phosphate group to active sites of enzymes is

a specific binding to enzymes in specific reaction.
 Substrate-level phosphorylation
The formation of ATP by phosphoryl group transfer from
substrate such as 1,3-biphosphoglycerate and
phosphoenolpyruvate.
O
O O P O- O O-
C O- C
Mg2+
CHOH + ADP CHOH + ATP
CH2OPO32- CH2OPO32-
Phosphoglycerate Kinase
1,3-biphosphoglycerate 3-phosphoglycerate

O O- O O-
C O C
Mg2+
C O P O- + ADP C O + ATP
CH2 O- Pyruvate Kinase CH2

Phosphoenolpyruvate Pyruvate
The net gain of energy from glycolysis
 Regulation of glycolysis

•Louis Pasteur discovered that both rate and total amount of

glucose consumption were many times greater under
anaerobic condition than aerobic condition.

•Later studies in muscles was clear that the ATP yields from
glycolysis under anaerobic condition is 2ATP, much lower than
the complete oxidation in aerobic condition (30 or 32ATP).

•18 times as much glucose must therefore be consumed in

anaerobic condition to yield same amount of ATP.

•The regulation of glycolysis is achieved by two glycolytic

enzymes, phosphofructokinase-1 and pyruvate kinase in ATP
(allosteric regulation).
 Trehalose Lactose

Sucrose

Feeder pathways
for Glycolysis
Degradation of glycogen and disaccharides
Glycogen of starch are degredrded by glycogen
phosphorylase
α-1-6 linkage

Glucose 1-phosphate Glycogen Phosphorylase

Transferase Activity of
Debranching Enzyme

α-1-6 Glucosidase Activity of

Oligo (α1-6) to (α1-4) glucantransferase Debrnaching Enzyme

Glycogen Phosphorylase
Fates of Pyruvate
Fermentation of pyruvate
 Fermentation of pyruvate to lactate

The fermentation of pyruvate is important to recycle the limited

NAD+ to degrade glucose during burst phase of working in
muscle.

2ATP
 Gluconeogenesis
•Biosynthesis of glucose is an absolute necessity in all
mammals.

•The brain and nervous system, erythrocyte, testes, renal

medulla, and embryonic tissue require glucose as a fuel
source.

•The formation of glucose from nonhexose precursor is

called gulconeogenesis.

•Gluconeogenesis is not occurred in every tissue types,

and occurred in glucogenic tissues, liver and kidney.
Gluconeogenesis
Three reactions of glycolysis are irreversible
and can not be used in gluconeogenesis.

•The conversion of glucose to glucose 6-phosphate by

hexokinase

•The phosphorylation of fructose 6-phosphate to

fructose 1,6-biphosphate by phosphofructokinase-1

•The conversion of phosphoenolpyruvate to pyruvate by

pyruvate kinase

“Gluconeogenesis requires bypass pathway of these

three reactions.’’
 Precursors for gluconeogenesis

I Glycerol Glycerol 3-phosophate Dihydroxyacetone

phosphate

II Carbon skeleton from

amino acid degradation

III Lactate Oxaloacetate Glucose

IV Pyruvate
Three reactions of glycolysis are irreversible
and can not be used in gluconeogenesis.
Gluconeogenesis
Gluconeogenesis
Alternative pathways from pyruvate to PEP
Transportation of oxaloacetate from mitochondria
into cytosol via malate-aspartate shuttle
 Cori cycle
Glucose
Blood Circulation Liver

Glucose Lactate

Muscle Glucose Blood Circulation

NAD+

NADH + H+
2 Pyruvate 2 Lactate
Muscle Muscle
 Reciprocal regulation of glycolysis and
gluconeogenesis
Glucose

2ADP + 2Pi 4ADP + 4GDP +4Pi

2ATP 4ATP + 4GTP

Pyruvate

•Both pathways required tight regulation.

•No net conversion, both glycolysis and gluconeogenesis
were allowed and the only net results is the utillization of
two ATPs and two GTPs (futile cycle).
 Reciprocal regulation of glycolysis and
gluconeogenesis
Regulatory enzymes
I Phosphofructokinase (PFK-1)
•Allosteric regulation by small molecules
-activators: AMP, fructose 2,6-bisphosphate
-inhibitors: ATP, citrate, H+ ion (prevention for
producing lactate) ATP ADP+Pi
•Fructose 2,6-bisphospshate PFK-2
is synthesized from
fructose 6-phosphate
Fructose 6-phosphate Fructose 2,6-bisphosphate

Pi
Fructose bisphosphatase 2
(FBPase2)
 Reciprocal regulation of glycolysis and
gluconeogenesis
Regulatory enzymes

PFK-2 is a bifunctional enzyme and both activities are catalyzed

by the same polypeptide.

Kinase Phosphatase

•PFK-2 is control by phosphorylation.

•Phosphorylated stage is active for phosphatase activity but
inactive for kinase activity.
 Reciprocal regulation of glycolysis and
gluconeogenesis

Lowered blood glucose Releasing of glucagon

Activation of Triggering cAMP cascade

protein kinase A

Acivation of FPBase 2
Phosphorylation of PFK-2
Inhibition of PFK-2

Lower glycolysis Lower fructose 2,6-bisphosphate

 Glycogen metabolism

•Glycogen is mainly stored in the liver and skeleton muscle.

•Muscle: source of energy during prolong muscle contraction
•Liver: maintaining blood glucose level

Glycogen degradation
•Enzyme glycogen phosphorylase (break α1-4 glycosidic
bond)
•Enzyme glycogen debranching enzyme (α 1-6 glycosidic bond)
•Product – glucose 1-phopshate
•Glucose 1-phosphate is converted into glucose 6-phosphate
by enzyme phosphoglucomutase, and enter to glycolysis.
 Glycogen metabolism
Glycogen synthesis
-Starting compound: UDP-glucose
-Enzyme: glycogen synthase (use UDP-glucose)
-Glycogen synthase require primer compose of complex
between glucose and protein (glycogenin)

UTP + glucose 1-phosphate UDP-glucose + PPi

UDP-glucose pyrophosphorylase

Branching enzyme
-Enzyme: amylo (1-4→1-6) transglycosylase creates α1-6
bond
 Glycogen metabolism

Regulatory enzymes for glycogen metabolism

I Glycogen phosphorylase
II Glycogen synthase (require high energy compound, UTP)

Both enzymes are required tight regulation in prevention of

futile cycle with no net conversion.

Glycogen

Pi UMP + PiPi

UTP
glucose 1-phosphate
 Glycogen metabolism

Hormonal regulation

Epinephrine Activate
Norepinephrine Adenylate cyclase
Glucagon
ATP cAMP

cAMP-dependent cAMP-dependent
protein kinase (inactive) protein kinase (active)

Phosphorylate target proteins

(Phosphorylase kinase)
 Glycogen metabolism

Epinephrine
Norepinephrine
Glucagon ATP ADP
Phosphorylase kinase

Phosphorylase kinase

phosphorylase b phosphorylase a
(inactive) (active)

Protein phosphatase I
Pi
 Glycogen metabolism
Hormonal regulation
Insulin (from β-cell of the pancreas)
-lower blood glucose after feeding and stimulate glycogen
synthesis

Insulin-responsive protein kinase

phosphorylate phosphorylate

Protein phosphatase I Protein phosphatase

inhibit phosphorylase activate glycogen synthase

 Glycogen metabolism
Hormonal regulation
Insulin activates protein phosphatase.

Protein phosphatase Pi

glycogen synthase glycogen synthase

(inactive) (active)

Protein kinase
ADP ATP
 Pentose phosphate pathway
-Glucose 6-phosphate can be the intermediate of other
catabolic fates, pentose phosphate pathway or
phosphogluconate pathway or hexose monophosphate
pathway.
-This pathway leads to produce specialized products needed
by the cell.
-Specialized products produced by pentose phosphate
pathway

10. NADPH for biosynthesis of fatty acids and steroids

11. Pentose (ribose 5-phosphate) for nucleic acid synthesis
 Pentose phosphate pathway

Glucose

Glucose 6-phosphate
Oxidation Oxidation
Pyruvate Pentose Phosphate Pathway

Citric Acid Cycle Ribose 5-phosphate NADPH

(Pentose)

Biosynthesis of Biosynthesis of
Nucleic Acid Fatty Acid and Steroid
Pentose phosphate pathway
Role of NADPH and glutathione in protecting cell
membrane
Glucose 6-phosphate dehydrogenase
deficiency (G6PD)
 Tricarboxylic acid cycle (TCA Cycle)
or
The citric acid cycle
Respiration
Broader sense: an physiological aspect referring
to a multicellular organism’s uptake of O2 and
release CO2
More narrow sense: a molecular process by
which cells consume O2 and produce CO2
(Cellular respiration).

•The TCA cycle is a center of metabolism in with degradative

pathways leading in and anabolic pathways leading out.
•The pathway of further oxidation of pyruvate into CO2, and
free energy is conserved in the reduced electron carriers,
NADH, FADH2 and ATP.
Oxidation of proteins, lipids and
carbohydrates into three stages
of cellular respiration.
 Oxidation of pyruvate into acetyl-CoA

CoA-SH NAD+ NADH

O O- O S CoA
C TPP, Lipoate C
FAD
C O C O + CO2
CH3 Pyruvate dehydrogenase CH3
complex
Pyruvate Acetyl-CoA

-The overall reaction is called oxidative decarboxylation

(irreversible).
-The carboxyl group is removed from pyruvate as a molecule
of CO2.
-The enzyme complex is composed of three enzymes.
 The citric acid cycle

-1948, Eugene Kennedy and Albert Lehninger showed that

reactions of the citric acid cycle in eukaryotic cells take place
in mitochondria.

- They also found that electron transfer and ATP synthesis by

oxidative phosphorylation occurs in inner membrane of
mitochondria.

- In most prokaryotic cells, the enzymes catalyzing reactions in

the citric acid cycle are in cytosol and plasma membrane play
a role analogous to that of the inner membrane of mitochondria
in ATP synthesis.
TCA cycle
 Products from single turn of the TCA Cycle
The GTP formed by succinyl-CoA synthetase can donate
phosphoryl group to ADP form ATP.
Mg2+
GTP + ADP GDP + ATP
Nucleoside diphosphate kinase
 Glycolysis

TCA cycle
Intermediates in citric acid cycle are important
biosynthetic precursors.
The citric acid cycle serves in both catabolic and anabolic
processes, amphibolic pathway.
 The reactions that replenish TCA intermediates.
As intermediates of the citric acid cycle are removed to serve
as biosynthetic precursors, they are replenished by
anaplerotic reaction.
 Pyruvate carboxylase is an enzyme important
to anaplerotic reaction.
-The most important anaplerotic reaction in mammalian kidney and liver
is the reversible carboxylation of pyruvate and CO2 to form oxaloacetate.

-Pyruvate carboxylase is a regulatory enzyme, which is controlled by

allosteric regulation, and is activated by excess amount of acetyl-CoA.

-The pyruvate carboxylase reaction requires the vitamin biotin, which is

prosthetic group of the enzyme.

-Biotin acts as a specialized one-carbon group in the most oxidized form,

CO2.

-Sources of biotin; many foods and intestinal bacteria

-The deficiency is rare, generally occurring when large quantities of raw

eggs are consumed (avidin in egg whites bind tightly to biotin preventing
for intestinal absorption).
 Oxidative phosphorylation

•1948, Eugene Kennedy and Albert Lehninger found that the

site of oxidative phosphorylation is occurred in mitochondria.

•The outer membrane of mitochondria: permeable to small

molecules (Mr< 5000) and ions by passing through
transmembrane proteins.

•The inner membrane is impermeable to most small molecules

and ions, including proton (H+) but it can be transported by
specific transporters.

•The inner membrane contains the component for ATP synthase.

Mitochondria
 Universal electron acceptors

-The electrons from catabolic pathways are kept in form of

NADH, NADPH, FADH2 and FMNH2 (oxidized forms, NAD,
NADP, FAD and FMN).
-Both NADH and NADPH can not cross the inner
mitochondrial membrane, but electrons can be shuttled
across indirectly.
-Flavoproteins: proteins contain FAD or FMN as a cofactor.
The FAD or FMN binds tightly to protein, sometimes
covalently attached to protein.
-The electron carriers will release energy in electron
transport chain.
 Summary of the flow electrons and protons

Higher [H+]

Lower [H+]

Energy of electron transfer is conserved in a proton gradient.

Energy from electron transfer is conserved in a
proton gradient (proton-motive force).

Proton-motive force provides

conserved energy in two forms.
• Chemical potential energy
From different concentration in
two separated sites.
2) Electrical potential energy
from separation of charge.

The free-energy change from electrochemical gradient by ion pump is

∆G = RT ln(C2 / C1 ) + ZF∆E
= 2.3RT∆pH + ZF∆E
Z = absolute value electrical charge (+1 for P+)
F = Faraday constant
 ATP synthesis
I Peter Mitchell proposed chemiosmotic model for ATP
synthesis mechanism.
II The electrochemical potential (proton-motive force) drives
the synthesis of ATP as protons flow passively back into the
matrix.

FADH2 = 2 ATP

NADH = 3 ATP
 The shuttle of cytosolic NADH reducing
equivalent into mitochondrial matrix

I Inner membrane of mitochondria is not permeable to NADH.

II Special shuttle systems carry reducing equivalents from
cytosolic NADH into mitochondria matrix by an indirect route.

Shuttle systems

7) Malate-aspartate shuttle system: Liver, Kidney

and heart muscle
9) Glycerol 3-phosphate shuttle system: Brain and muscle
Malate-aspartate shuttle system

NADH = 3 ATP
Glycerol 3-phosphate shuttle system

FADH2 = 2 ATP