Far Eastern University — Nicanor Reyes Medical Foundation

School of Medicine
Biochemistry A – Carbohydrate Metabolism I & II
Remedios P. Santos, M.D.

METABOLIC MAP - Major fuel of most organisms & occupies a central position in
metabolism
- Relatively rich in potential energy; the complete oxidation of glucose
to CO2 & H2O proceeds with a standard free-energy change of -2840
kJ/mol
- By storing glucose as glycogen, a cell can stockpile large quantities
of hexose units while maintaining a relatively low cytosolic
osmolarity
 When energy demands suddenly increase, glucose can be
release quickly from glycogen & used to produce ATP
either aerobically or anaerobically

WHY IS GLUCOSE CONSIDERED AS THE MOST IMPORTANT

CARBOHYDRATE?
- The characteristic sugar of blood and tissue fluids is glucose.
- It is as glucose that the bulk of dietary CHO is absorbed into the
bloodstream or into which it is converted in the liver.
- It is from glucose that all other CHO in the body can be formed.
 Ribose in nucleic acids, galactose in milk, glycoproteins,
proteoglycans
- Glucose is a major fuel of the tissues of mammals and a universal
fuel of the fetus.
- The metabolism of many of the protein amino acids proceeds by the
glucose pathway, and some of the products of glucose metabolism
are utilized by the body for the synthesis of amino acids.
 Pyruvate can be converted to alanine
 The LIVER plays a central role in the metabolism of the
different carbohydrates. The same way it also plays a
central role in the metabolism of lipids and proteins.

FOOD CARBOHYDRATES
1. 60% - starches and dextrins
2. 30% - sucrose
CARBOHYDRATES OR SACCHARIDES (CHO) 3. 5% - lactose
- From Greek word “Saccharon” meaning sugar 4. 5% - other sugars
- Essential components of all living organism 5. Cellulose – there is no hydrolase in man that could digest
- Most abundant biomolecule on earth cellulose
- As partially reduced molecules, CHO can be oxidized to yield energy
to drive metabolic processes (acting as energy-storage molecules) DIGESTION OF CARBOHYDRATES
- CHO polymers (AKA glycans) serve as structural and protective I. Digestion in the mouth
elements in the cell walls of bacteria and plants and in the  Enzyme: salivary amylase or ptyalin
connective tissue of animals  Usually insignificant
- Other CHO polymers:
 Bonds acted upon: 1,4-glycosidic linkages of amylopectin
o May lubricate skeletal joint and participate in recognition
and amylose
and adhesion between cells
II. Digestion in the stomach
o Derivatives of sugars are also found in a number of
biological molecules including: antibiotics, coenzymes and  No enzymatic digestion
nucleic acid  Limited acid hydrolysis
- Glycobiology – study of the structure and function, in health and III. Digestion in the Small Intestines
diseases of various types of CHO  Enzymes:
i. Pancreatic amylase or amylopsin – same action as
MULTIPLE ROLES OF CARBOHYDRATES IN ALL FORMS OF LIFE: salivary amylase
1. Serve as energy stores, fuels and metabolic intermediates ii. Oligosaccharidases – from the brush border of the
 It is the preferred source of energy by the organism—gives small intestines; contained in the intestinal juice
of 4kcal/g or 32 to 34 moles of ATP per mole of glucose secreted by the glands of Brunner and of Lieberkühn
2. Ribose and deoxyribose sugars form part of the structural iii. -limit dextrinase – acts on limit dextrins
framework of DNA and RNA iv. Sucrase – acts on sucrose
3. Polysaccharides are structural elements in the cell walls of bacteria v. Lactase – acts on lactose
and plants, and in the exoskeletons of arthropods vi. Maltase – acts on maltose
 Cellulose – main constituent of plant cell walls; most vii. Isomaltase or (1,6) glucosidase – spilts -1,6-
abundant organic compound in the biosphere (1,4 glycosidic bonds
linkage)  End product of CHO digestion: Monosaccharides (GGF)
4. Linked to many proteins and lipids forming glycoconjugates which i. Glucose
can serve as recognition sites for hormones, antigen specificity, etc. ii. Galactose
5. Generate reducing equivalents in the form of NADPH which is iii. Fructose
essential in the biosynthesis of important cellular components like
lipids, steroids, DNA, etc. *No more enzymatic digestion in the Large Intestines 
D-GLUCOSE ABSORPTION OF CARBOHYDRATES

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A. Actively absorbed – needs carrier and energy 1. To produce energy in the form of ATP
1. Galactose – most rapidly absorbed 2. Intermediates formed can be converted to other substances
2. Glucose like amino acids, fatty acids, etc.
B. Facilitated transport – needs carrier only; no energy
1. D-fructose Tissues that depend on Glycolysis as their
2. D-mannose major mechanism for ATP production
Rate of monosaccharide absorption RBC
(From the most rapidly absorbed to the least rapidly absorbed) Cornea, lens, regions of the Lacks mitochondria
Galactose  Glucose  Fructose  Mannose  Pentose retina
They are almost totally
FATES OF INGESTED GLUCOSE Kidney medulla, testis, leukocytes dependent on glycolysis as
50% Converted to energy through glycolysis and white muscle fibers source of ATP because they have
30-40% Converted to fat relatively few mitochondria
10% Converted to glycogen for storage It has an absolute need for
glucose and processes most of it
FIRST REACTION UNDERGONE BY GLUCOSE ONCE INSIDE THE CELL via glycolysis. Approx. 120g
Brain ofglucose is used by the adult
human brain each day in order to
meet its extraordinary need for
ATP.
Adapted for aerobic
hexokinase performance, has relatively poor
glucokinase
Heart muscle glycolytic ability, and poor
survival under conditions of
ischemia
***Combined, the tissues that are dependent primarily on glycolysis for
ATP production consume about 40g of glucose per day in the normal
human adult.
Differences between Hexokinase and Glucokinase ***In fast growing cancer cells, glycolysis proceeds at a much higher rate
Hexokinase Glucokinase than is required by the Kreb’s cycle—more pyruvate produced than can be
Low Km for glucose, so it can High Km for glucose, so it can metabolized. Excessive production of lactate  Lactic acidosis
convert glucose in the function only when the
micromolar range to glucose-6- concentration of glucose is TWO PHASES OF GLYCOLYSIS
phosphate in the millimolar relatively high ***This is according to Lehninger Principles of Biochemistry (6th Ed)
range which Dra. Santos said is more accurate.
Inhibited by high levels of Not inhibited by glucose-6- 1. Energy Investment Phase – also called Priming Stage
glucose-6-phosphate (feedback phosphate a. Preparatory Stage
inhibition) Glucose  Fructose 1,6-biphosphate
Actos on other hexoses such as Acts on glucose only b. Splitting stage
fructose, mannose, etc. Fructose 1,6-biphosphate  DHAP + glyrealdehyde-3-PO4
2. Energy Generation Phase – also called Energy Recovery Phase
FATES OF GLUCOSE-6-PHOSPHATE 1. Oxidoreduction stage – Phosphorylation stage
1. Conversion to glycogen Glyceraldehyde-3-PO4  Pyruvate or Lactate
2. Enter glycolysis
3. Conversion to fatty acids and cholesterol GLYCOLYSIS INTERMEDIATES
4. Conversion to blood glucose All intermediates of glycolysis between glucose and pyruvate are
5. Oxidative degradation to CO2 – energy formation phosphorylated compounds. Their phosphate group appears to have 3
6. Degradation via HMP shunt functions:
7. Enters Uronic Acid Pathway 1. Their phosphate groups of the glycolytic intermediates also
serve as binding or recognition groups in the formation of
GLYCOLYSIS enzyme-substrate complexes.
- Major pathway for glucose utilization 2. They provide each intermediate with a polar, negatively
- Found in all cells charged group.
- Plays a key role in energy metabolism by providing a significant 3. The phosphate groups function in the conservation of energy
portion of the free energy used by most organisms and by preparing since they ultimately become the terminal phosphate groups of
glucose and other compounds for further oxidative degradation ATP in the course of glycolysis.
- A unique pathway since it can utilize oxygen if available (aerobic) or
it can function in the absence of oxygen (anaerobic) KINDS OF REACTIONS THAT OCCUR IN GLYCOLYSIS
- The pathway used by all cells of the body to extract part of the 1. Phosphoryl transfer – a phosphoryl group is transferred from ATP to
chemical energy inherent in the glucose molecule a glycolytic intermediate or vice versa
- It sets the stage for the complete oxidation of glucose to H2O and a. Example: Glucose + ATP  Glucose-6-Phosphate + ADP
CO2 2. Phosphoryl shift – a phosphoryl group is shifted within a molecule
- It also provides the main pathway for the metabolism of fructose from one oxygen atom to another
and galactose derived from the diet a. Example: 3-phosphoglycerate  2-phosphoglycerate
- It is a sequence of reactions that converts glucose to pyruvate and 3. Isomerization – a ketose is converted into an aldose or vise versa
lactate with concomitant production of ATP a. Example: Glucose-6-phosphate  Fructose-6-phosphate
- It takes place in the cytosol 4. Dehydration – a molecule of water is eliminated
- It is basically an exergonic process a. Example: 2-Phoshoglycerate  Phosphoenolpyruvate -
- All intermediates between glucose and pyruvate are phosphorylated H2O
- The word glycolysis is derived from the Greek words glykos meaning 5. Aldol cleavage – a carbon-carbon bond is split in a reversal of an aldol
“sweet” and lysis meaning “splitting” or “loosing” condensation
FUNCTIONS OF GLYCOLYSIS

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 a. Example: Fructose-1,6-biphosphate  Dihydroxyacetone - Glucose is activated for subsequent reaction by its phosphorylation
PO4 + Glyceraldehyde-3-PO4 at C-6 to yield glucose 6-phosphate with 1 ATP as the phosphoryl
donor
THE GLYCOLITC PATHWAY - Type of reaction: Irreversible Phosphorylation
- Enzymes: hexokinase and glucokinase
- Recall: Kinases are enzymes that catalyze the transfer of the terminal
phosphoryl group from ATP to an acceptor molecule. In Hexokinase,
the acceptor is a hexose (normally D-glucose). It can also catalyze
the phosphorylation of other common hexoses: D-fructose and D-
mannose, in some tissues.
glucokinase - Hexokinase, like many other kinases, requires Mg2+
- Hexokinase undergoes profound change in shape, an induced fit,
when it binds glucose. This movement brings bound ATP closer to a
molecule of glucose also bound to the enzyme and blocks the access
of water (from the solvent), which might otherwise enter the active
site and attack (hydrolyze) the phosphoanhydride bonds of ATP.

Second Reaction: Conversion of Glucose 6-Phosphate to Fructose 6-

Phosphate

- Enzyme: Phosphohexose isomerase (phosphoglucose isomerase)

- Type of reaction: Reversible Isomeration of glucose 6-phosphate, an
aldose, to fructose 6-phosphate, a ketose
- This reaction involves an enediol intermediate
- This reaction proceeds readily in either direction

Third Reaction: Phosphorylation of Fructose 6-Phosphate to Fructose 1,6-

Biphosphate

ENERGY INVESTMENT PHASE

- Priming stage
- Requires ATP
- Two molecules of ATP are invested and the hexose chain is cleaved - Enzyme: Phosphofructokinase-1 (PFK-1)
into two triose phosphates - Type of reaction: Irreversible phosphorylation
- PFK-1 catalyzes the transfer of a phosphoryl group from ATP to
fructose 6-phosphate to yield fructose 1,6-biphosphate
Preparatory Stage
- Good to know: Compounds that contain two phosphate or
First Reaction: Phosphorylation of Glucose
phosphoryl groups attached at different positions in the molecule
are name biphosphates or biphospho compounds.
- This reaction is irreversible under cellular conditions
- The first committed step in the glycolytic pathway
- Glucose 6-phosphate and fructose 6-phosphate have other possible
glucokinase
fates but fructose 1,6-biphosphate is targeted for glycolysis
- RATE LIMITING/CONTROL POINT

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 Splitting Stage - The aldehyde group (R-CHO) of glyceraldehyde 3-phosphate is
Fourth Reaction: Cleavage of Fructose 1,6-Biphosphate oxidized, not to a free carboxyl group but to a carboxylic acid
anhydride with phosphoric acid – acyl phosphate.
- Acyl phosphate – has a very high standard free energy of hydrolysis

- Enzyme: Aldolase
- Type of reaction: Reversible Aldol Condensation
- A.K.A. Splitting Stage
Fifth Reaction: Interconversion of the Triose Phosphates
- NADH + H is released in this reaction
- When NADH is released, it should be reverted back to NAD. The
reducing equivalent (NADH) is given off to the Electron Transport
Chain (ETC)
- Note: The amount of NAD+ in a cell is far smaller that the amount of
glucose metabolized in a few minutes. Glycolysis would soon come
to a halt if the NADH formed in this step of glycolysis were not
continuously reoxidized and recycled.
- Glycolysis takes place in the Cytosol while the ETC is in the
mitochondria, therefore, a SHUTTLE SYSTEM is needed

Shuttle Systems:
1. Malate-Aspartate Shuttle System
- Enzyme: Triose phosphate isomerase  Enters COMPLEX I (giving you a P:O ratio of 2.5)
- Only one of the two triose phosphates formed by aldolase,  2.5 ATPs are produced once your reducing equivalents
glyceraldehyde 3-phosphate, can be directly degraded in the enter ETC through this system
subsequent steps of glycolysis
- The other product, dihydroxyacetone phosphate, is rapidly and
reversibly converted to glyceraldehyde 3-phosphate by the fifth
enzyme: triose phosphate isomerase
- Reaction: Isomerization
- The two halves of glucose have both yielded glyceraldehyde 3-
phosphate
- This reaction completes the preparatory phase of glycolysis

PAYOFF PHASE
- Oxidative conversion of glyceraldehyde 3-phosphate to pyruvate
and the coupled formation of ATP and NADH

Oxidoreduction-Phosphorylation Phase
- Recall: DHAP (dihydroxyacetone) and Glyceraldehyde 3-phosphate
are interchangeable. Therefore, from the 5th Reaction, the
succeeding steps will produce two similar pathways and will produce
x2 products per step (one for each pathway).
- The conversion of two molecules of glyceraldehyde 3-phosphate to
two molecules of pyruvate is accompanied by the formation of four 2. Glycerol Phosphate Shuttle System
molecules of ATP from ADP. However, the net yield of ATP per  Enters COMPLEX II bypassing the COMPLEX I giving off a P:O
molecule of glucose degraded is only twom because two ATP were ratio of 1.5
invested in the preparatory phase of glycolysis to phosphorylate the  1.5 ATPs are produced once your reducing equivalents
two ends of the hexose molecule. enters Complex II of the ETC
Sixth Reaction: Oxidation of Glyceraldehyde 3-Phosphate to 1,3-
Biphosphoglycerate

- First step in Payoff Phase

- Enzyme: glyceraldehyde 3-phosphate dehydrogenase
- Type of reaction: Oxidative Phosphorylation
- This is the first of the two energy-conserving reactions of glycolysis
that eventually lead to the formation of ATP.

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 Eight Reaction: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

- Enzyme: Phosphoglycerate mutase

- Type of reaction: Phosphoryl shift
- Reversible

Ninth Reaction: Dehydration of 2-Phosphoglycerate to

Phosphoenolpyruvate

- Oxidative Phosphorylation – production of energy (ATP) through

the ETC

How many ATPs are produced through Oxidative Phosphorylation?

 Malate Aspartate Shuttle System
o 2.5 ATP multiplied by (2) NADH = 5 ATPS
- 1 from Glyceraldehyde produced from the cleavage of
- Enzyme: Enolase
Fructose 1,6-biphosphate in the Fourth Reaction
- Type of reaction: Dehydration
- 1 from Glyceraldehyde converted from the DHAP in the
- Reversible
Fifth Reaction
 Glyceraldehyde Phosphate Shuttle System
Tenth Reaction: Transfer of Phosphoryl Group from Phosphoenolpyruvate
- 1.5 ATP multiplied by (2) FADH2 = 3 ATPS to ADP
- Source of NADH is same as from the Malate Aspartate - The last step in glycolysis
Shuttle System
- Enzyme: Pyruvate kinase
- Type of reaction: Substrate-level phosphorylation
Seventh Reaction: Phosphoryl Transfer from 1,3-Biphosphoglycerate to - Irreversible reaction
ADP
- Enzyme: Phosphoglycerate kinase
- Type of reaction: Phosphoryl transfer/Substrate level of
Phosphorylation (Didn’t go inside ETC but produced ATP)
- Reversible

- Note: 6th and 7th Steps of glycolysis together constitute an energy-

coupling process in which 1,3-biphosphoglycerate is the common
intermediate. It is formed in the first reaction (which would be
endergonic in isolation_ and its acyl phosphate group is transferred
to ADP in the second reaction (which is strongly exergonic).

- Sum of the 6th and 7th reactions:

- The overall reaction is exergonic.

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 SUMMARY OF THE GLYCOLYTIC PATHWAY
Substrate Enzyme Product Type of Reaction Features Inhibitors
ENERGY INVESTMENT PHASE
Preparatory Stage
Hexokinase or Glucose 6- - 1 ATP is used 2-
1st Glucose Phosphorylation
Glucokinase Phosphate (G6P) - Irreversible Deoxyglucose
Phosphohexose
- Reversible
Glucose 6- isomerase Fructose 6-
2nd Isomerization - G6P (aldose) to F6P (ketose) or
Phosphate (Phosphoglucose Phosphate (F6P)
vice versa
isomerase)
- 1 ATP is used to phosphorylate
F6P
- Irreversible
Fructose 6- Phosphofructokinase- Fructose 1,6- - Rate limiting/Control point
3rd Phosphorylation
Phosphate 1 (PFK1) Biphosphate - 1st committed step in glycolytic
pathway
- Fructose 1,6-Biphosphate is
targeted for glycolysis
Splitting Stage
- Reversible
Dihydroxyacetone
- A.K.A. Splitting stage
phosphate
Fructose 1,6- - The 2 sugars that are produced
4th Aldolase + Aldol Condensation
Biphosphate from halving Fructose 1,6-
Glyceraldehyde 3-
Biphosphate have 3 carbons
phosphate
each
- Reversible
- Only Glyceraldehyde can be
Dihydroxyacetone Triose phosphate Glyceraldehyde 3- degraded in the subsequent
5th Isomerization
phosphate isomerase phosphate steps of glycolysis that’s why
DHAP needs to be converted to
GA3P
ENERGY GENERATION PHASE/PAYOFF PHASE
Oxidoreduction-Phosphorylation Stage
- 1st of the 2 energy-conserving
reactions
- NADH + H is released
- H+ will go to ETC
- NADH will be reoxidized back
to NAD in order for the Sulfhydryl
Glyceraldehyde 3- 1,3- pathway to continue reagents (ex.
Glyceraldehyde 3- Oxidative
6th phosphate Biphosphoglycerat Iodoacetate)
Phosphate Phosphorylation
dehydrogenase e # of ATPs is produced if: - inhibit
- Malate-Aspartate Shuttle GA3P DH
System (Aerobic) – 5 ATPs
(2.5 x 2NADH = 5ATPs)
- Glycerol Phosphate Shuttle
System (Aerobic) – 3ATPs
(1.5 x 2FADH = 3ATPs)
Phosphoryl
1,3- 3-
Phosphoglycerate transfer (Substrate - Production of ATP outside ETC
7th Biphosphoglycerate phosphoglycerate
kinase level of - 2 ATPs produced
+ ADP + ADP
Phosphorylation)
3- Phosphoglycerate 2- Phosphoryl
8th - Reversible
Phosphoglycerate mutase Phosphoglycerate Transfer
2- - Reversible
9th Enolase Phosphoenolpyruvate Dehydration Fluoride
phosphoglycerate - 1 mol of H2O is removed
Phosphoenolpyruvate - Irreversible
Substrate-level
10th + Pyruvate kinase Pyruvate + ATP - Last step in glycolysis
phosphorylation
ADP - 2 ATPs produced
Lactic Acid
Pyruvate Lactate
Dehydrogenase

- Irreversible Steps: Steps 1, 3 and 10

- ATP consumption: Steps 1, and 3
- ATP generation: Steps 6, 7, 10
- Note: All reversible steps in glycolysis are control points.
- Rate limiting step/committed step
o Once you passed by this step, you are sure that the pathway will proceed down to the last step.
o Major control point of the pathway

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 ENZYMATIC CONTROL OF GLYCOLYSIS FATES OF PYRUVIC ACID
1. Phosphofructokinase-1 (PFK) 1. Reversible reduction to lactate
 Inhibited by 2. Conversion back to glucose
o ATP 3. Formation of oxaloacetate or malate
o Citrate 4. Transamination to form alanine
o H+ ions 5. Oxidative decarboxylation to Acetyl CoA
o Cyclic AMP (cAMP)
o ATP/AMP ratio
 Stimulated by
o ADP
o AMP
o  ATP/AMP ratio
2. Hexokinase – inhibited by Glucose-6-Phosphate
3. Pyruvate kinase
 Inhibited by
o ATP
o Alanine
o Fatty acids CELLULAR RESPIRATION
o Acetyl CoA - Some cells obtain energy (ATP) by fermentation—breaking down
 Stimulated by glucose in the absence of oxygen
o Fructose 1,6-Biphosphate - For most eukaryotic cells and many bacteria which live under
Note: aerobic conditions and
- Under anaerobic conditions, NADH is supposed to accumulate oxidize their organic fuels
because of the absence of Electron Transport Chain (ETC) BUT to carbon dioxide and
due to the reduction of pyruvate to lactate, NAD regeneration water, glycolysis is the first
occurs sustaining the continued operation of glycolysis EVEN stage in the complete
under Anaerobic Conditions. oxidation of glucose
- Pyruvate produced by
glycolysis is further
oxidized to H2O & CO2
- Respiration – the aerobic
phase of catabolism
- Cellular respiration – the
molecular processes by
which cells consume O2 &
produce CO2
- Most important function of glycolysis is production of energy. - Stages of Cellular
o So 1 factor that controls it is the energy level of the body respiration:
o ↑ energy: ↑ ATP/ADP ratio & ↑ ATP/AMP ratio 1. Oxidation of
o ↑ ATP/ADP & ↑ ATP/AMP means that you have a high level glucose, fatty
of 
energy acids, and some
o If ↑ energy, ↓ ATP/ADP & ↓ ATP/AMP you will INHIBIT amino acids to
glycolysis 
by inhibiting the control enzymes Acetyl CoA
o Glycolysis is inhibited because of the high level of energy so 2. Citric Acid Cycle
you don’t need to produce anymore AT 3. Oxidative
o Energy level determines whether you stimulate or inhibit phosphorylation
glycolysis

INHIBITORS OF THE GLYCOLYTIC PATHWAY PYRUVATE DEHYDROGENASE COMPLEX

1. 2-Deoxyglucose - Needs to take place for Pyruvate to enter the Krebs Cycle
 Inhibits the reaction catalyzed by hexokinase
2. Sulfhydryl reagents
 Ex: iodoacetate
 Inhibit glyceraldehyde-3-phosphate dehydrogenase
 Glyceraldehyde-3-PO4 dehydrogenase has a sulfhydryl
group that combines with Glyceraldehyde-3-PO4 to give a
thiohemiacetal Sulfhydryl groups are usually mercury-
containing compounds or alkylating compounds which
readily combines with the sulfhydryl group of
Glyceraldehyde-3-PO4 dehydrogenase preventing the
formation of thiohemiacetal
3. Fluoride
 A potent inhibitor of Enolase
 Mg2+ and Pi form an ionic complex with Fluoride ion, which
is responsible for inhibition of enolase by interfering with
binding of its substrate (Mg2+ 2-Phosphoglycerate) 

4. Pentavalent arsenic or arsenate Enzymes Prosthetic Groups
 Arsenic poisoning inhibits those enzymes which require Pyruvate dehydrogenase Thiamine pyrophosphate (TPP) or
Lipoic Acid as coenzymes like pyruvate dehydrogenase & Vit B1
-ketoglutarate dehydrogenase (not part of glycolysis) Dihydrolipoyl transacetylase Lipoamide and CoA or Vit B5
Dihydrolipoyl dehydrogenase FAD or Vit B2 & NAD or Vit B3

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 PYRUVATE DEHYDROGENASE
- First one to act
- Absence of Thiamine (which is a coenzyme of PDH)  No Reaction
will take place
Note:
- Once you produce pyruvate, it enters the mitochondria so it’s
conversion to acetyl CoA is inside the mitochondria.
o NAD will accept the H+ to become NADH and this will
enter ETC
o There is no need to a shuttle system anymore because
this takes place in the mitochondria
o Right away NADH will produce 2.5 ATP per NADH but
remember there are 2 pyruvate, so it becomes 2.5 x 2
= 5 ATP
- So for this step, you produce 5 moles ATP thru the ETC ✓ Acetyl
CoA enters the Kreb’s cycle for final conversion to energy. It must
enter the Kreb’s.
PYRUVATE DEHYDROGENASE REGULATORS
1. Endproduct inhibition
 Dehydrogenase is inhibited by its products, Acetyl Coa and
NADH
2. Covalent modification
 Pyruvate dehydrogenase is regulated by phosphorylation
by a kinase of 3 serine residues on the pyruvate
dehydrogenase component, resulting in decreased
activity
 By dephosphorylation by a phosphatase that cause an
increase in activity
 The kinase is activated by increases in the ATD/ADP ratio
and acetyl CoA/CoA ratio, and NADH/NAD+ ratio
PDH COMPLEX REQUIRES FIVE COENZYMES
1. Thiamine pyrophosphate (TPP)
2. Flavin adenine dinucleotide (FAD)
3. Nicotinamide adenine dinucleotide (NAD)
4. Coenzyme A (CoASH)
5. Lipoate 
 - Much of the lactate produced in
FOUR VITAMINS THAT ARE COMPONENTS OF PDH COMPLEX
1. Vitamin B1 (Thiamine) in Thiamine Pyrophosphate (TPP)
2. Vitamin B2 (Riboflavin) in FAD
3. Vitamin B3 (Niacin) in NAD
4. Vitamin B5 (Pantothenic acid) in CoA 
 - Accumulation = muscle cramps
If you lack Vitamin B, you will not be able to convert the Pyruvate to
Acetyl CoA and it cannot enter the Krebs Cycle.
Major manifestation of vitamin B deficiencies:
1. Weakness because glucose can’t enter Krebs Cycle
2. Severe muscle weakness
 Thiamin is the first coenzyme you are going to use
 So if thiamine is deficient  can’t convert pyruvate to
CoA  no Krebs Cycle  manifest with severe muscle
weakness
 Muscle affected isn’t only the skeletal muscles
 It also affects cardiac muscles, so it could be serious
3. Lactic acidosis
 Because pyruvate cannot be converted to acetyl CoA,
it will now be channeled in lactic acid production
 This can also kill the patient, that’s why it’s very
important that pyruvate must be converted into acetyl
CoA
Beriberi
 caused by Vitamin B1 deficiency
 Shoshin beriberi
o Patient will go into cardiac failure
o Fulminating (very serious) type
o Can lead to heart failure
INHIBITORS OF PYRUVATE DEHYDROGENASE
1. Arsenite and Mercuric Ions react with the –SH groups of lipoic acid
(lipoate) & inhibit pyruvate dehydrogenase
2. Thiamine Deficiency – nutritionally deprived alcoholics are thiamine-
deficient and may develop potentially fatal pyruvic and lactic
acidosis
INBORN ERRORS OF METABOLISM ASSOCIATED WITH GLYCOLYSIS
1. Inherited Aldolase A Deficiency
2. Inherited Pyruvate Kinase Deficiency
3. G6PD Deficiency
 Aldolase is a cleavage enzyme while pyruvate kinase is the
enzyme for the last step of aerobic glycolysis. G6PD is under the
HMP shunt.
4. Muscle Phosphofructokinase Deficiency
 Exercise capacity of patient is low particularly on high CHO diet
 No energy production since this is the most important rate-
limiting enzyme
 Energy production is low  Exercise capacity is low
 Phosphofructokinase is the rate limiting enzyme of the
glycolytic pathway. Without it, glycolysis will stop  energy
production will be very low  exercise capacity will also be very
low
5. Inherited Pyruvate Dehydrogenase Deficiency
 Due to defects in one or more of the components of the
enzyme complex
 Manifested by lactic acidosis particularly after a glucose load
CORI CYCLE OR LACTIC ACID CYCLE
Fate of Lactic Acid/Lactate in Anaerobic
Glycolysis
- Lactate represents a sort of dead
end for anaerobic glucose
metabolism
- Lactate can either be exported from
the cell or converted back to
pyruvate
skeletal muscle cells is carried by the
blood to the liver where it is used to
synthesize glucose (Cori Cycle)
Lactic acid should not be allowed to accumulate in the muscles
- Must mobilize this (through massage) so that it does not
cause cramps
- Massaging will facilitate the entry of lactate back to the
blood, then to the liver
- Once in the liver, it will be converted back to glucose
through glucogenesis and it can go back to the blood again
to be used by the muscles
Lactate is being recycled in the liver:
- There are two processes involved:
1. Glycolysis
2. Glycogenesis
ACETYL COA
- Biological acetylating agent in the synthesis of compounds such as
acetylcholine, acetylsulfanilamide, N-acetyl sugars & the acetylated
N-termini of peptide chains
- Most important fate of Pyruvate is to be converted to Acetyl CoA
(only way to Krebs Cycle)
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- Central metabolite which can also come from: o Anabolic part is when you use the intermediates to synthesize
1. Glucose – produced if pyruvate is converted to acetyl CoA other substances
2. Beta-oxidation of fatty acids - Location: mitochondria besides the ETC
3. Catabolism of amino acids o So when NADH is released, you no longer need a shuttle
- Acetyl CoA comes from all the food that can be consumed system; NADH will automatically produce 2.5 ATP
- 80% - must enter the Krebs Cycle which requires insulin (has a - All enzymes are located in the mitochondrion, so all substrates,
permissive effect on the Krebs Cycle) including NAD+ * GDP must be generated in the mitochondria OR
- 20% - converted to ketone bodies, cholesterol, bile acids, steroid can be transported into the mitochondria FROM the cytosol
hormones, prostaglandins, TAG - Carbon atoms of the two molecules of CO2 produced in one round
of the cycle ARE NOT the two carbon atoms of the acetyl group that
Case: Uncontrolled Diabetes Mellitus began the round
- Majority of Acetyl CoA cannot enter Krebs Cycle o These acetyl carbon atoms are lost in the subsequent rounds
- Will be channeled towards ketone body production of the cycle
o Before 20% o However, the net effect of each round of the cycle is the
o Now 20% + 80% oxidation of one acetyl group of 2 CO2
o This is why one of the dreaded complications of
DM is ketoacidosis and it kills
- Can lead to production of more cholesterol
o Consequences is atherosclerosis
o Atherosclerosis can bring about MI, cerebral
hemorrhage and stroke
- Complication of DM can be partly due to the fact that acetyl
CoA cannot enter the Krebs since it is more channeled
towards ketone body or more cholesterol production
o If cholesterol blocks retinal artery  blindness
(hence why blindness is a complication of DM)
o If it blocks the renal arteries  renal failure (once
the kidneys are the ones affected in DM, you are
sure to die); even dialysis will not reverse the
effects; kidneys are the last ones to fail among
diabetics

INSULIN HAS A PERMISSIVE EFFECT ON THE KREBS CYCLE

- Insulin is needed in order for Acetyl CoA to enter Krebs Cycle
- Among diabetics who lack insulin, instead of 80% of Acetyl CoA
entering the Krebs Cycle, it is diverted to ketone bodies formation
 Ketoacidosis (one of the complications of diabetic patients that
can kill them instantly)
- Acetyl CoA can also proceed to cholesterol production in the
absence of insulin leading to high cholesterol  Atherosclerosis 
blocks arteries  cerebral stroke, coronary MI, retinal blindness or
renal failure

KREBS CYCLE/TRICARBOXYLIC ACID (TCA) CYCLE

- Other names: Tricarboxylic Acid Cycle/Citric Acid Cycle
- Final common pathway of metabolism REACTION INVOLVED IN THE KREB’S CYCLE
o Maximum energy production happens when glucose First Reaction: Formation of Citrate
enters both glycolysis and Krebs
- Discovered by Hans Krebs in 1937
- Considered as the “hub” of cellular metabolism since it accounts for
the major portion of CHO, FA and AA oxidation
- One complete round of the cycle yields
o 2 molecules of CO2
o 3 NADH
o 1 FADH
o 1 “high-energy” compound (GTP/ATP)
- Condensation of acetyl-CoA with oxaloacetate to for citrate
FUNCTIONS OF THE KREBS CYCLE - Enzyme: Citrate synthase
1. Major degradative pathway for the generation of ATP - Type of reaction: Claisen condensation
2. Provides intermediates for biosynthesis (amphibolic pathway) - Citrate synthase is formerly known as “condensing enzyme”
a. Alpha-ketoglutarate – precursor of glutamic acid - Rate-limiting step/Committed step
b. Oxaloacetate – precursor of aspartic acid
c. Citrate – precursor of extra mitochondrial acetyl CoA Second Reaction: Formation of Isocitrate via cis-Aconitate
for fatty acid biosynthesis – through the ATP-citrate
lyase reaction
d. Citrate + ATP + CoA  Acetyl CoA + Oxaloacetate +
ADP + Pi
e. Succinyl CoA – for the synthesis of heme
- Amphibolic
o Means both a catabolic (degradation) and anabolic (synthesis
of substances)
o Catabolic part is the generation of ATP

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- Enzyme: Aconitase (formerly known as aconitate hydratase)
- Type of reaction: Dehydration/Rehydration Seventh Reaction: Hydration of Fumarate to Malate
- Reversible transformation of citrate to isocitrate through the
intermediary formation of the tricarboxylic acid cis-aconitate
- Aconitase can promote the reversible addition of H2O to the double
bond if enzyme-bound cis-aconitate in two different ways, one
leading to citrate and the other to isocitrate
- d Aconitase contains an iron-sulfur center, which acts both in the
binding of the substrate at the active site and in the catalytic
addition or removal of H2O.
- In iron-depleted cells, aconitase loses its iron-sulfur center and
acquires a new role in the regulation of iron homeostasis.
- Aconitase is one of many enzymes known to “moonlight” in a second - Enzyme: fumarase
role. - Type of reaction: hydration
- Gains H2O
Third Reaction: Oxidation of Isocitrate to -Ketoglutarate and CO2
Eight Reaction: Oxidation of Malate to Oxaloacetate

- Enzyme: L-malate dehydrogenase

- Enzyme: Isocitrate dehydrogenase - Type of reaction: dehydrogenation
- Type of reaction: oxidative decarboxylation - Release of NADH yielding 2.5 ATPs
- Isocitrate, the substrate, loses one carbon by oxidative
decarboxylation SUMMARY:
- Releases 1 NADH (or NADPH) & 1 CO2 - Dehydration: Steps 2 & 7
- No shuttle is needed since the Krebs Cycle takes place in the - Rehydration: Step 2
mitochondria - Release of CO2: Steps 3 & 4
- Upon release of 1 NADH, it enters Complex I of ETC producing 2.5 - Release of NADH: Steps 3, ,4 & 8
ATPs - Release of FADH2: Step 6
- ATP Consumption: NONE
Fourth Reaction: Oxidation of -Ketoglutarate to Succinyl-CoA and CO2 - ATP Generation:
o Steps 3, 4 & 8 though NADH formation (7.5 ATPs)
o Step 6 through FADH2 formation (1.5 ATPs)
o Step 5 through GTP formation (1 ATP)
For every turn of the Krebs Cycle
- Oxidative phosphorylation (NADH & FADH2  enters the ETC)
3NADH: 3 x 2.5 = 7.5 ATPs
- Enzyme: -Ketoglutarate dehydrogenase complex 1FADH2: 1 x 1.5 = 1.5 ATPs
- Type of reaction: Oxidative decarboxylation 9 ATPs
- Releases 1 NADH & 1 CO2 - 1 substrate level: 1 ATP
- Upon release of 1 NADH, it enters Complex I of ETC producing 2.5 Total: 10 ATPs
ATPs
CONTROL POINTS IN THE KREBS CYCLE
Fifth Reaction: Conversion of Succinyl-CoA to Succinate
Positive
Control Points Rxn Catalyzed Inhibitors
Modulator
Acetyl CoA + ATP, Citrate,
Oxaloacetate Succinyl-CoA, ADP
 Citrate NADH
Citrate
Synthase - Rate of this reaction is largely dependent on
- Enzyme: Succinyl-CoA synthase or succinic thiokinase the availability od Acetyl CoA & Oxaloacetate
- Type of reaction: substrate-level phosphorylation - High concentration of Succinyl CoA competes
- Reversible with Acetyl CoA inhibiting Citrate Synthase
- 1 ATP is released even though you do not enter ETC Isocitrate 
Isocitrate - ATP Ca++, ADP
Sixth Reaction: Oxidation of Succinate to Fumarate ketoglutarate
Succinyl CoA
-
ketoglutarate NADH+
- Ca++
 succinyl
ketoglutarate High energy
CoA
dehydrogenase charge
complex - Inhibited by Arsenite causing -ketoglutarate
- Enzyme: succinate dehydrogenase to accumulate just like Pyruvate
- Type of reaction: dehydrogenation Dehydrogenase
- Release of FADH2 Irreversible formation of acetyl CoA from Pyruvate
- Upon release of 1 FADH, it enters Complex II of ETC producing 1.5
ATPs

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 SUMMARY OF THE KREBS CYCLE
Substrate Enzyme Product Type of Reaction Features Inhibitors
ATP, Citrate,
- Irreversible, rate limiting
Acetyl CoA + Claisen Succinyl-CoA,
1st Citrate synthase Citrate - Methyl group of acetyl-CoA
Oxaloacetate condensation NADH, High
converted to methylene in citrate
energy
- Reversible
Dehydration/ - —OH group of citrate repositioned
2nd Citrate Aconitase Isocitrate
Hydration in isocitrate to set up
decarboxylation in next step
- Irreversible, rate limiting
- —OH group oxidized to carbonyl,
which in turn facilitates
decarboxylation by stabilizing
carbanion formed on adjacent
Isocitrate - Oxidative carbon
3rd Isocitrate ATP
dehydrogenase Ketoglutarate decarboxylation - Releases 1 NADH (or 1 NADPH) & 1
CO2
- No shuttle needed because it takes
place INSIDE the mitochondria
- Upon entering Complex I of ETC,
NADH produces 2.5 ATPs
Succinyl-CoA,
- Irreversible, rate limiting
NADH, High
-Ketoglutarate Oxidative - Releases 1 NADH & 1 CO2
4th -Ketoglutarate Succinyl-CoA energy charge,
DH complex decarboxylation - Upon entering Complex I of ETC,
arsenate (acc
NADH produces 2.5 ATPs
to Inah)
- Reversible
Succinyl-CoA Substrate-level
5th Succinyl-CoA Succinate - 1 ATP is released even though it
synthase phosphorylation
does not enter ETC
- Reversible
Malonate
- Release of 1 FADH  enters
6th Succinate Succinate DH Fumarate Dehydrogenation (competitive
Complex II of ETC  produces 1.5 inhibitor)
ATPs
- Reversible
7th Fumarate Fumarase L-Malate Hydration
- Gains H20
L-malate - Reversible
8th L-Malate Oxaloacetate Dehydrogenation
dehydrogenase - Release of 1 NADH

SUMMARY Per turn of the Krebs cycle

- Dehydration: Steps 2 - Release 2 mol water from
- Rehydration: Step 2 & 7 - Aconite step
- Release of CO2: Steps 3 & 4 - Fumarate step
- Release of NADH: Steps 3, 4 & 8 - Also release 2 mol CO2 from:
- Release of FADH2: Step 6 - Isocitrate dehydrogenase step
- ATP Consumption: NONE - α-ketoglutarate step
- ATP Generation:
o Steps 3, 4 & 8 though NADH formation (7.5 ATPs) SUMMARY
o Step 6 through FADH2 formation (1.5 ATPs) Per turn of the Krebs cycle you produce:
o Step 5 through GTP formation (1 ATP) - 10mol ATP

For every turn of the Krebs Cycle - 2mol water

- Oxidative phosphorylation (NADH & FADH2  enters the ETC) - 2mol CO2

3NADH: 3 x 2.5 = 7.5 ATPs - 3 NADH (contributes 7.5 ATP)
1FADH2: 1 x 1.5 = 1.5 ATPs - 1 FADH2 (contributes 1.5 ATP) 

9 ATPs
- 1 substrate level: 1 ATP REGULATION OF KREBS CYCLE
Total: 10 ATPs
- For every turn of Krebs, a total of 10 ATPs are produced. The rate of the Krebs Cycle depends upon two factors:
- From 1 mole of glucose, 20 ATPs are produced in TCA (from 2 1. Energy requirements of the cell
Acetyl-CoA that came from 2 Pyruvates in Glycolysis). 2. Requirement of the cell for carbon precursors

There are 3 control points and the rate limiting step. “control point” HIGH ENERGY ( ATP) CHARGE DECREASES activities of:
- If you want to stimulate the pathway, you don’t stimulate all - Citrate synthase
enzymes only the control enzymes - Isocitrate dehydrogenase
- If you want to inhibit the pathway, you only inhibit the control - a-ketoglutarate dehydrogenase
enzymes
Result: DECREASED ACTIVITY OF KREB’S CYCLE.
Note:

Glycolysis and Krebs are partners, so when you stimulate glycolysis,
it’s automatic that Krebs is also stimulated. GENERATION OF HIGH ENERGY BONDS IN THE CATABOLISM OF GLUCOSE
- High energy charge = ↓ glycolysis → ↓ Kreb’s cycle
- Low energy charge = ↑ glycolysis → ↑ Krebs cycle

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 # of ATP All major members of the Krebs Cycle from Citrate to
Method of
Pathway Rxn Catalyzed by produced Oxaloacetate are potentially glucogenic since they can
energy
involved what enzyme per mole of give rise to a net production of glucose in the liver &
production
glucose kidneys
Glyceraldehyde-3-respiratory chain Phosphoenolpyruvate carboxykinase (PEPCK)
PO4 dehydrogenase oxidation of 2 5  Key enzyme that facilitates the net transfer out of the
NADH Krebs cycle into the main pathway of Gluconeogenesis
Glycolysis Phosphoglycerate Oxidation at 2. Transamination & Deamination
2
kinase substrate-level  These reactions produce pyruvate from alanine,
Pyruvate kinase Oxidation at oxaloacetate from aspartic acid & -ketoglutarate from
2
substrate-level glutamic acid 

GROSS 9  These reactions are reversible so the Krebs Cycle also
ATP consumed in investment phase (hexokinase and serves as a source of carbon skeletons for the synthesis of
-2
phosphofructokinase) the above nonessential amino acids 

NET 7 3. Fatty Acid Synthesis 

Respiratory  Acetyl CoA is the major building block for long chain fatty
PDH chain oxidation 5 acid synthesis in nonruminants
of 2 NADH  Since the enzymes responsible for fatty acid synthesis are
Isocitrate DH Respiratory extramitochondrial, the cell needs to transport acetyl CoA
chain oxidation 5 through the mitochondrial membrane which is
of 2 NADH impermeable to Acetyl CoA
-Ketoglutarate Respiratory  How is this accomplished?
chain oxidation 5 i. Acetyl CoA is converted to Citrate
of 2 NADH ii. Citrate is transported out of the Mitochondria
Succinate Oxidation at iii. Acetyl CoA is made available in the cytosol by
Krebs Cycle 2
thiokinase substrate level cleaving citrate
Malate Respiratory iv. catalyzed by the enzyme ATP-citrate lyase
dehydrogenase chain oxidation 5
of 2 NADH ANAPHLEROTIC REACTIONS/FILLING-UP REACTIONS
Succinate DH Respiratory - Replenish Citric Acid Cycle Intermediates
chain oxidation 3 - As intermediates of the TCA Cycle are removed to serve as
of 2 FADH biosynthetic precursors, they are replenished by Anaphlerotic
GROSS 25 Reactions
ATP CONSUMPTION 0 o Because the Krebs cycle is a very important pathway, your
NET 25 body sees to it that there are always substrates that are
Total ATP production per mole of glucose under available
32 o If you lack a substrate, there are reactions that can refill
aerobic condition (using malate aspartate shuttle)
them
In order for glucose to have a complete oxidation, it needs to go thru:
1. Glycolysis EXAMPLES OF ANAPHLEROTIC REACTIONS
2. PDH Complex 1. Formation of Malate FROM Pyruvate by Malaic Enzyme
3. Krebs Cycle o Pyruvate + CO + NADPH + H+ ⟷ Malate + NADP
RECAP!!! We’ve learned the ff: 2. Glutamate + Pyruvate ⟷ -Ketoglutarate + Alanine and
 Glycolysis occurs in the CYTOSOL producing 2 Pyruvate Aspartate + Pyruvate ⟷ oxaloacetate + Alanine
 2 Pyruvate enters the Mitochondria By: Transaminase
3. Enzymatic Carboxylation of Pyruvate to form Oxaloacetate
 2 Pyruvate is converted to 2 Acetyl CoA through the PDH
o Most important anaphlerotic reaction in the liver & kidney
Complex
 Major anaphlerotic reaction
 2 Acetyl CoA enters Krebs Cycle = 2 Cycle Occurs
o Production of oxaloacetate from pyruvate, ATP, CO2
o Requires biotin (vitamin B7) as coenzyme
Only activated
VITAMINS THAT PLAY IMPORTANT ROLES IN THE KREB’S CYCLE
if there is acetyl CoA which is a positive modulator of
1. Vitamin B2 / Riboflavin – FAD (Flavin adenine dinucleotide)
pyruvate carboxylase
 Cofactor in the a-ketoglutarate dehydrogenase complex
o Occurs in two steps:
 Cofactor of succinate dehydrogenase 

E-Biotin + ATP + CO2 + H2O ⟷ E-carboxybiotin + ADP + Pi
2. Niacin or nicotinic acid – NAD (Nicotinamide adenine dinucleotide)
E-carboxybiotin + Pyruvate ⟷ E-Biotin + Oxaloacetate .
 Coenzyme for isocitrate dehydrogenase, a-ketoglutarate
ATP + CO2 + Pyruvate + H2O ⟷ Oxaloacetate +ADP Pi 

dehydrogenase & malate dehydrogenase

 Catalyzed by pyruvate carboxylase
3. Vitamin B1 / Thiamine– Thiamine pyrophosphate or TPP
 Coenzyme for decarboxylation in the a-ketoglutarate
dehydrogenase complex 

4. Pantothenic Acid – part of Coenzyme A
 Cofactor attached to “active” carboxylic acid residues such as
Acetyl CoA & Succinyl CoA 

TCA CYCLE PLAYS A KEY ROLE IN THE FOLLOWING METABOLIC PATHWAYS:
1. Gluconeogenesis

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 GLUCOGENESIS
- Reverse of Glycolysis and Krebs
- HYPERglycemic pathway – activated when blood sugar level is high
- Includes all pathways and mechanisms responsible for converting
non carbohydrates to glucose or glycogen
- Pathway that takes place only in the LIVER & KIDNEYS
Relevance:
 Maintains blood sugar even if a person is not having adequate
CHO in the diet (during starvation)
 Normal blood sugar level depends on the method used:
o Floin Wu: 80-120mg/dL
o Glucometer: depends on the test strip
 Normal Blood Glucose Level: 70-110mg/dL
o Below 70mg/dL: Hypoglycemia
o Above 70mg/dL: Hyperglycemia
o If left untreated, Hypo and Hyper may cause death
o Hypoglycemia is more fatal than Hyperglycemia
o Diabetics may die in their sleep because of
Hypoglycemic Shock

IMPORTANCE OF GLUCONEOGENESIS
1.

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