BIOCHEMISTRY - VIII

CARBOHYDRATE METABOLISM
 Major Pathways of CHO Metabolism
• Glycolysis: Oxidation of glucose to pyruvate
(aerobic state) or lactate (anaerobic state)
• Krebs cycle: After oxidation of pyruvate to
acetyl CoA, acetyl CoA enters the Krebs
cycle for the aim of production of ATP.
• Gluconeogenesis: Formation of glucose from
noncarbohydrate sources.
• Hexose monophosphate shunt: Enables cells
to produce ribose-5-phosphate and NADPH.
• Glycogenesis: Synthesis of glycogen from
glucose, when glucose levels are high
• Glycogenolysis: Degradation of glycogen to
glucose when glucose in short supply.
• Stage 1: Digestion of Carbohydrates

In Stage 1, the digestion of carbohydrates:

• Begins in the mouth where salivary amylase breaks down
polysaccharides to smaller polysaccharides (dextrins), maltose, and
some glucose.
• Continues in the small intestine where pancreatic amylase
hydrolyzes dextrins to maltose and glucose.
• Hydrolyzes maltose, lactose, and sucrose to monosaccharides,
mostly glucose, which enter the bloodstream for transport to the
cells.
Digestion of Carbohydrates
 Stage 2: Glycolysis

• Is a metabolic pathway that

uses glucose, a digestion
product.
• Degrades six-carbon
glucose molecules to three-
carbon (pyruvate
molecules).
• Can be an anaerobic (no
oxygen) or aerobic process.
• All glycolysis reactions occur in the cytosol.
• Two phases of glycolysis:
• preparatory phase is the stage in which there is consumption of ATP
• ATP-generating phase

• The first five steps of the glycolysis reaction is known as the

preparatory or investment phase. This stage consumes energy to
convert the glucose molecule into two molecules three-carbon sugar
molecule.
• The Glycolytic Pathway

In glycolysis, a pathway with

10 reactions, each glucose
molecule is converted into two
pyruvate molecules.
1. The step one in glycolysis is phosphorylation. This step glucose is
phosphorylated by the enzyme hexokinases/glucokinases. In this
process, ATP molecule is consumed. A phosphate group from the ATP
is transferred to the glucose molecules to produce glucose-6-
phosphate. 
• Glucose (and other hexoses) are phosphorylated immediately
upon entry into the cell. Phosphorylation prevents transport of
glucose out of the cell and increases the reactivity of oxygen in
the resulting phosphate ester.
• Several isoenzymes of hexokinase with different Km values
for glucose are located in different tissues. Brain hexokinase
has a particularly low Km for glucose.
• The major enzyme for phosphorylating glucose in liver is
glucokinase.
2. The second stage of glycolysis is an isomerization reaction. In this
reaction the glucose-6-phosphate is rearranged into fructose-6-
phosphate by the enzyme glucose phosphate isomerase. This is a
reversible reaction under normal conditions of the cell. 
3. In the third step of glycolysis is a phosphorylation reaction -
irreversible . In this step the enzyme phosphofructokinase(PFK-1) is
transfers phosphate group to form fructose 1,6-bisphosphate. Another
ATP molecule is used in this step.
4. Cleavage of fructose-1,6-bisphosphate. Stage 1 of glycolysis ends with
the cleavage of fructose-1,6-bisphosphate into two three-carbon
molecules:glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone
phosphate (DHAP). This reaction is an aldol cleavage, hence the name of
the enzyme: aldolase.
5. The interconversion of glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate. Of the two products of the aldolase
reaction, only G-3-P serves as a substrate for the next reaction in
glycolysis. To prevent the loss of the other three-carbon unit from the
glycolytic pathway,triose phosphate isomerase catalyzes the reversible
conversion of DHAP to G-3-P:
GLICOLYSIS – the
energy-consuming phase
GLICOLYSIS – the ATP-
generating phase
6. Oxidation of glyceraldehyde-3-phosphate. During reaction 6 of
glycolysis, G-3-P undergoes oxidation and phosphorylation. The product,
glycerate-1,3-bisphosphate, contains a high-energy phosphoanhydride bond,
which may be used in the next reaction to generate ATP. This complex
process is catalyzed by glyceraldehyde-3-phosphate dehydrogenase. NAD+
is the electron acceptor → is converted to NADH

7. Phosphoryl group transfer. 1, 3-bis-phosphoglycerate is converted to 3-

phosphoglycerate by the enzyme 1, 3-bis-phosphoglycerate kinase. Here
one molecule of ATP is formed.
• is an example of a substrate-level phosphorylation (without oxygen
participation).
• synthesis of ATP is endergonic, it requires an energy source.

• In substrate level phosphorylations, ATP is produced by the transfer of a

phosphoryl group from a substrate with a high phosphoryl transfer potential
(glycerate- 1,3-bisphosphate) to produce a compound with a lower transfer
potential (ATP) and therefore G ˂ 0.
Because two molecules of glycerate-1,3-bisphosphate are formed for every
glucose molecule, this reaction produces two ATP molecules, and the
investment of phosphate bond energy is recovered.
8. 3-phosphoglycerate is isomerized to 2-phosphoglycerate by
shifting the phosphate group from 3rd to 2nd carbon atom. The
enzyme is phosphoglucomutase.

9. 2-phosphoglycerate is converted to phosphoenol pyruvate by the

enzyme enolase. One water molecule is removed. A high energy
phosphate bond is produced. This enzyme requires Mg++and
inhibited by fluoride.
10. Phosphoenol pyruvate is dephosphorylated to pyruvate, by
pyruvate kinase. One molecule of ATP is generated. This step is
irreversible.

• This is the second substrate-level phosphorylation reaction in

glycolysis
 The Fates of Pyruvate
• When oxygen is available (left), aerobic organisms completely oxidize pyruvate to CO2 and H2O. In
the absence of oxygen, pyruvate can be converted to several types of reduced molecules. In others
(e.g., muscle cells), homolactic fermentation occurs in which lactate is the only organic product
(right).
• Aerobic conditions: NADH is re-oxidized in the respiratory chain
→ the end product of glycolysis is pyruvate
• Anaerobic conditions: NADH is re-oxidized by donating its
electrons to pyruvate, which is reduced to lactate in the reaction
catalyzed by lactate dehydrogenase (LDH) → the end product of
anaerobic glycolysis is lactate (which accumulates)
• The NADH produced during the conversion of glyceraldehyde-3-
phosphate to glycerate-1,3-bisphosphate is oxidized when pyruvate is
converted to lactate. This process allows the cell to continue producing
ATP under anaerobic conditions as long as glucose is available.

Recycling of NADH During Anaerobic

Glycolysis
Energy yield from glycolysis
Phase I: 2 ATP are consumed
- hexokinase / glucokinase reaction
- phosphofructokinase reaction
•Phase II: 4 ATP are generated by substrate-level phosphorylation (2 ATP for each molecule
of triose phoshpate)
- phosphoglycerate kinase reaction
- pyruvate kinase reaction
• ANAEROBIC GLYCOLYSIS:
glucose + 2ADP + 2Pi → 2 lactate + 2ATP + 2H2O
4 ATP – 2 ATP = 2 ATP
• AEROBIC GLYCOLYSIS
glucose + 2NAD+ + 2ADP + 2Pi → 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O
GLUCOSE-DEPENDENT TISSUES

• Brain – substantial energy consumption → satisfied mostly from

glucose (can NOT obtain energy from fatty acids)
• Red blood cells – does not contain mitochondria → cannot degrade
other energy sources (which are oxidized in the mitochondria and
involve oxidative phosphorylation)
• Skeletal muscle undergoing a heavy and prolonged exercise – has high
energy needs compared to a decreased oxygen supply → an important
part of ATP is obtained in anaerobic glycolysis
Regulation of glycolysis
• The rate at which the glycolytic pathway operates in a cell is
directly controlled primarily by the kinetic properties of its
hexokinase isoenzymes and the allosteric regulation of the enzymes
that catalyze the three irreversible reactions:
• hexokinase,
• PFK-1 (catalyzes the rate-limiting step),
• pyruvate kinase.
 Glucokinase – induced by insulin

Piruvate kinase - covalent regulation (in the liver) - glucagon phosphorylation inactivation
Regulation of PFK-1 by F-2,6-BP in the liver
Regulation of pyruvate kinase
METABOLISM OF PYRUVATE
• Pyruvate, a key molecule in metabolism of human and its fate
differs depending upon presence and absence of oxygen.

• It is the end product of glycolysis and is eventually transported into

mitochondria as a major energy and participates in the TCA cycle.

• In the glycolysis, glucose is converted into two molecules of

pyruvate with the generation of ATP. However, if reactions stops at
pyruvate, due to imbalance redox, it would not proceed for long.
Anaerobic conditions – pyruvate lactate

Aerobic conditions – pyruvate:

• Oxidative decarboxylation to acetyl-CoA
(Pyruvate dehydrogenase complex)
• Oxidation of acetyl-CoA to CO2
(Krebs cycle)
• In the presence of oxygen, molecules like glucose and other sugars,
fatty acids, and most amino acids are eventually oxidized to CO2
and H2O via the TC A cycle and the respiratory chain.

• In the matrix of the mitochondria, first pyruvate is converted to

Acetyl-CoA by the enzyme pyruvate dehydrogenase complex
(PDC) because former cannot enter the TCA cycle.
PDC holds a key position in connecting the glycolytic and
Oxidative pathway of the TCA cycle.

This catalysis is sequential process which involves the oxidative

decarboxylation of pyruvate and the formation of acetyl-CoA, CO2 and
NADH (H+). This reaction needs five co-factors namely Co-A, TPP, lipoate,
FAD and NAD+.

PDC are made up of several copies of three catalytic enzymes namely:

- pyruvate dehydrogenase (E1),
- dihydrolipoyl transacetilase (E2), and
- dihydrolipoyl dehydrogenase (E3)
TPP (thiamine pyrophosphate)( B1-vit)

Thiamine
Lipoic acid
Coenzyme A (CoA-SH)
Regulation of Pyr. Dehydrogenase Complex
• Allosteric
Acetyl-CoA and NADH inhibits E1
(are the end products of pyruvate dehydrogenase reaction)
• Covalent regulation:

• PDH kinase catalyzes the phosphorylation of E1 → leads to enzyme inactivation

- it is activated by increased amounts of acetyl-CoA, NADH and ATP

• PDH phosphatase catalyzes the dephosphorylation of E1 → leads to enzyme activation

- it is activated by Ca2+ and insulin (in the adipose tissue)

acetyl-CoA, NADH and ATP are formed when fatty acids degradation is active → pyruvate
degradation is slowed down
• Acetyl CoA plays a central role in metabolism.In fact most
energy generating metabolic pathways of the cell
eventually produce it.

• It can be formed from carbohydrate, fat and protein.

• It is also the starting point for the synthesis of fats, steroids
and ketone bodies.
• Its oxidation provides energy for many tissues.
KREBS CYCLE
(TCA)
• The citric acid cycle is the final common pathway for the oxidation of fuel
molecules amino acids, fatty acids, and carbohydrates. Most fuel molecules
enter the cycle as acetyl coenzyme A.
• A cyclical series of eight reaction that oxidize one molecule of acetyl CoA
completely to two molecules of CO2, generating energy, either directly as ATP
or in the form of reducing equivalents (NADH or FADH2).
• The cycle is aerobic; the absence or deficiency of oxygen leads to total or
partial inhibition of the cycle.
Location:
• All mammalian cells that contain mitochondria (i.e. not red blood cells)
Site:
• All the enzymes of TCA cycle are located in the mitochondrial matrix.
Function of TCA cycle
1.The TCA cycle provides final common pathway for the oxidation of
carbohydrate, fat and protein.
2. The main function of the cycle is the production of energy, either,
directly as ATP or as the reducing equivalents, NADH or FADH2, which
are oxidized by ETC.
3. The cycle provides substrates for the ETC.
4. The cycle is also a source of biosynthetic precursors, for example,
porphyrin is synthesized from succinyl CoA, and amino acids are
synthesized from oxaloacetate and α- ketoglutarate.
5. Some of the cycle intermediates also exerts regulatory effects on other
pathways; for example, citrate inhibits PFK-1 in glycolysis.
 • isocytrate dehydrogenase(NAD+ dependent)
• α-ketoglutarate dehydrogenase (NAD+dependent)
dehydrogenation • succinate dehydrogenase (FAD dependent)
(DH) reactions • malate dehydrogenase (NAD+ dependent)

• citrate synthase
irreversible • Isocytrate dehydrogenase
reactions • α-ketoglutarate dehydrogenase

substrate-level • succinate thiokinase

phosphorylation
Amphibolic nature of the citric acid cycle
• In aerobic organisms, the citric acid cycle is an amphibolic pathway,
one that serves in both catabolic and anabolic processes.

• Besides its role in the oxidative catabolism of carbohydrates, fatty

acids, and amino acids, the cycle provides precursors for many
biosynthetic pathways through reactions that served the same purpose
in anaerobic ancestors.

• TCA cycle is actively involved in gluconeogenesis, transamination and

deamination.
• The most important synthetic (anabolic) reactions connected with
TCA cycle are:
1. Oxaloacetate and α-ketoglutarate, respectively, serve as precursors
for the synthesis of aspartate and glutamate which, in turn, are required
for the synthesis of other non-essential amino acids, purines and
pyrimidines.

2. Succinyl CoA is used for the synthesis of porphyrins and heme.

3. Mitochondrial citrate is transported to the cytosol, where it is cleaved

to provide acetyl CoA for the biosynthesis of fatty acids, sterols etc.
Anaplerosis or Anaplerotic reactions
• As intermediates of the citric acid cycle are removed to serve as
biosynthetic precursors, they are replenished by anaplerotic
reactions.
• The reactions concerned to replenish or to fill up the intermediates
of citric acid cycle are called anaplerotic reactions or anaplerosis
(Greek : fill up).
• Under normal circumstances, the reactions by which cycle
intermediates are siphoned off into other pathways and those by
which they are replenished are in dynamic balance, so that the
concentrations of the citric acid cycle intermediates remain almost
constant
 The salient features of important anaplerotic
reactions are
1. Pyruvate carboxylase catalyses the conversion of pyruvate to oxaloacetate. This is an
ATP dependent carboxylation reaction.
• Pyruvate + CO2 + ATP → Oxaloacetate + ADP + Pi
2. Pyruvate is converted to malate by NADP+ dependent malate dehydrogenase (malic
enzyme).
• Pyruvate + CO2 + NADPH + H+ → Malate + NADP+ + H2O
3. Transamination is a process where in an amino acid transfers its amino group to a
keto acid and itself gets converted to a keto acid. The formation of α- ketoglutarate and
oxaloacetate occurs by this mechanism.
4. of α-ketoglutarate can also be synthesized from glutamate by glutamate
dehydrogenase action.
• Energy yield of Krebs cycle: 12 ATP (catabolic aspect of TCA)
- 3 NADH (3 x 3ATP)
- 1 FADH2 (2 ATP)
- molecules formed during the cycle → oxidized in the respiratory chain → synthesis of 11 ATP in
the process of oxidative phosphorylation
- 1 ATP is synthesized through substrate-level phosphorylation
Energy yield of the total aerobic degradation of glucose
• Regulation of TCA cycle
Three regulatory enzyme namely:
• Citrate synthase,
• Isocitrate dehydrogenase and
• α-Ketoglutarate dehydrogenase regulate citric acid cycle.

The rate-limiting step – isocitrate dehydrogenase

• The inner mitochondrial membrane is impermeable to NADH
• Reducing equivalents collected in glycolysis (in the form of
NADH) have to be transported in the mitochondria (for oxidation in
the respiratory chain) with the help of shuttle systems
• These are possible due to the existence of dehydrogenases with
double localization: in the cytosol and in the mitochondria
Malate
shuttle

Glycerol-3-P
shuttle
 GLUCONEOGENESIS
• the formation of new glucose molecules from
noncarbohydrate precursors, occurs primarily in the liver.

Precursor molecules:
• lactate,
• pyruvate,
• glycerol, and certain -keto acids (molecules derived from amino
acids).
The precursors for
gluconeogenesis
1. Gluconeogenesis from lactate
• Lactate is provided by anaerobic glycolysis in exercising skeletal
muscle and red blood cells → Cori cycle
• GNG from lactate takes place as a reversal of glycolysis – but NOT
on an entirely identical pathway
• The reversible reactions of glycolysis are run in opposite direction,
in GNG
• The 3 irreversible steps of glycolysis (highly exergonic)
enzymes specific for GNG
• Synthesis of PEP. PEP synthesis from pyruvate requires two
enzymes:pyruvate carboxylase and PEP carboxykinase. Pyruvate carboxylase,
found within mitochondria, converts pyruvate to oxaloacetate (OAA):The
transfer of CO2 to form the product OAA is mediated by the coenzyme biotin ,
which is covalently bound within the enzyme’s active site.
Transport of oxaloacetate
from mitochondria to the
cytosol
• OAA is then decarboxylated and phosphorylated by PEP carboxykinase in a
reaction driven by the hydrolysis of guanosine triphosphate (GTP):
Conversion of fructose-1,6-bisphosphate to fructose-6-
phosphate.The irreversible PFK-1–catalyzed reaction in glycolysis is
bypassed by fructose-1,6-bisphosphatase:
• Formation of glucose from glucose-6-phosphate. Glucose-6-
phosphatase,found only in liver and kidney, catalyzes the
irreversible hydrolysis of glucose-6-phosphate to form glucose and
Pi. Glucose is subsequently released into the blood.
• Gluconeogenesis is an energy-consuming process. Instead of
generating ATP (as in glycolysis), gluconeogenesis requires the
hydrolysis of six high energy phosphate bonds.

EQUATION OF GLUCONEOGENESIS FROM LACTATE

• 2 lactate + 4 ATP + 2 GTP + 4 H2O → glucose + 4 ADP + 2 GDP + 6 Pi
 2. Gluconeogenesis from glycerol

• Glycerol, a product of fat metabolism in adipose tissue, is transported to

the liver in the blood and then converted to glycerol-3-phosphate by
glycerol kinase. Oxidation of glycerol-3-phosphate to form DHAP
occurs when cytoplasm NAD concentration is relatively high.
• Fatty acids are partially taken up by the liver → oxidized
to acetyl-CoA
• To be transformed into glucose, acetyl-CoA should be
converted to pyruvate
• But the opposite reaction, catalyzed by pyruvate
dehydrogenase, is irreversible → acetyl-CoA (and thus
fatty acids with an even number of C atoms) CANNOT be
converted to glucose
 3. Gluconeogenesis from amino acids

18 out of 20 amino acids can

be converted to glucose (they
are glucogenic amino acids) –
exceptions are leucine and
lysine
• alanine is perhaps the most important – In the liver, alanine is converted
to pyruvate in a transamination reaction catalyzed by GPT
• Glucose is sent back to the muscle to be used as an energy source for
contraction (glucose – alanine cycle)
Gluconeogenesis Regulation
1. Allosteric regulation
• Pyruvate carboxylase – activated by acetyl-CoA
• Fructose-1,6-bisphosphatase – inhibited by F-2,6-BP and AMP

2. Hormonal control through induction/repression

• Glucagon, epinephrine and cortisol – induce the 4 key-enzymes, especially
PEPCK
• Insulin – represses them
• After a meal:
- high insulin/glucagon ratio → high amount of F-2,6-BP →
PFK-1 is activated and FBPase is inhibited → glycolysis is
accelerated and GNG is slowed down

• During fasting:
- low insulin/glucagon ratio → low amount of F-2,6-BP →
FBPase is activated and PFK-1 is inhibited → GNG is accelerated
and glycolysis is slowed down
References

•David L. Nelson, Michael M. Cox. Lehninger Principles of Biochemistry. 5th edition, 2008.
•Colleen Smith, Allan D. Marks, Michael Lieberman. Marks’ Basic Medical Biochemistry: A
Clinical Approach. 2nd edition, 2004.
•Robert K. Murray, Darryl K. Granner, Peter A. Mayes, Victor W. Rodwell. Harper’s
Illustrated Biochemistry. 27th edition, 2006.
•Pamela C. Champe, Richard A. Harvey, Denise R. Ferrier. Lippincott’s Illustrated Reviews
– Biochemistry. 4th edition, 2007.
•Reginald H. Garrett, Charles M. Grisham. Biochemistry. 2nd edition, 1999.
•Mary K. Campbell, Shawn O. Farrell. Biochemistry. 6th edition, 2007.