IMPORTANT CARBOXYLATE IONS IN

METABOLIC PATHWAYS
• Structurally-related polyfunctional
carboxylate ions
• 5 polyfunctional carboxylate ions serve as
substrates for some enzymes in the
metabolic reactions.
• 5 structures related to 2 simple carboxylic
acids: SUCCINIC ACID and GLUTARIC ACID
 NO. OF C DERIVATIVES
ATOMS

SUCCINIC 4 HYDROXY DERIVATIVE:

MALIC ACID (MALATE)
KETO DERIVATIVE:
OXALOACETIC ACID (OXALOACETATE)
UNSATURATED DERIVATIVE:
FUMARIC ACID (FUMARATE)
(ALL HAVE 4 CARBON ATOMS, CHARGE OF 2-)
GLUTARIC 5 KETO DERIVATIVE:
α-KETOGLUTARIC ACID (α-KETOGLUTARATE)
(5 CARBON ATOMS, CHARGE OF 2-)
CARBOXYHYDROXY DERIVATIVE:
CITRIC ACID (CITRATE)
(6 CARBON ATOMS, CHARGE OF 3-)
 HIGH ENERGY PHOSPHATE
COMPOUNDS
• Several phosphate-containing compounds
found in metabolic pathways are known as
high energy compounds.
• A HIGH ENERGY COMPOUND is a compound
that has a greater free energy of hydrolysis
than that of a typical compound.
 HIGH ENERGY PHOSPHATE
COMPOUNDS
• The free energy is the amount of energy
released by a chemical reaction that is actually
available for further use at a given temperature
and pressure.
• In reality, the energy released in a chemical
reaction is divisible in two parts. One part, lost
as heat, is not available for further use. The
other part, the free energy is available for
further use to “drive” reactions that require
energy.
 HIGH ENERGY PHOSPHATE
COMPOUNDS
• High energy compounds differ from other
compounds in that they contain one or more
very reactive bonds, often called as strained
bonds.
• The energy required to break these strained
bonds during hydrolysis is less than that
generally required to break a chemical bond.
 HIGH ENERGY PHOSPHATE
COMPOUNDS
• Consequently, the balance between the
energy needed to break bonds in the
reactants and that released by bond
formation in the products is such that more
than the typical amount of free energy is
released during the hydrolysis reaction.
 HIGH ENERGY PHOSPHATE
COMPOUNDS
• These HEP compounds obey the normal rules for
chemical bonding. The only difference between
such compounds and other compounds in the
presence of one or more strained bonds.
• The breaking of such bonds requires lower-than-
normal amounts of energy.
 Overview of Biochemical Energy
Production
• The energy needed to run the human body is
obtained from the ingested food through a
multiple process that involves several,
different catabolic pathways.
• There are four general stages in the
biochemical energy production process, and
numerous reactions are associated with each
stage.
 Overview of Biochemical Energy
Production
• Stage 1-Digestion
• Stage 2-Acetyl Group Formation
• Stage 3- Citric acid cycle/KC/TCA
• Stage 4-ETC and OP
 Overview of Biochemical Energy
Production
• The reactions in stages 3 and 4 are the same for all
types of foods. These reactions constitutes the
common metabolic pathway.
• The common metabolic pathway is the sum total
of the biochemical reactions of the CAC, ETC and
OP.
• The reactions for stages 1 and 2 of biochemical
energy production differ for all different types of
foodstuffs.
 THE CITRIC ACID CYCLE
• The citric acid cycle — also known as the
tricarboxylic acid cycle (TCA cycle), or the
Krebs cycle
• is a series of chemical reactions used by all
aerobic organisms to generate energy through
the oxidation of acetate derived from
carbohydrates, fats, and proteins into carbon
dioxide and chemical energy in the form of
(ATP).
 THE CITRIC ACID CYCLE
• In addition, the cycle provides precursors of
certain amino acids as well as the reducing
agent NADH that is used in numerous other
biochemical reactions.
• Its central importance to many biochemical
pathways suggests that it was one of the
earliest established components of cellular
metabolism and may have originated
abiogenically.
 THE CITRIC ACID CYCLE
• The name of this metabolic pathway is
derived from citric acid (a type of tricarboxylic
acid) that is consumed and then regenerated
by this sequence of reactions to complete the
cycle.
• In addition, the cycle consumes acetate (in
the form of acetyl-CoA) and water, reduces
NAD+ to NADH, and produces carbon dioxide
as a waste byproduct.
 THE CITRIC ACID CYCLE
• The NADH generated by the TCA cycle is fed into the
oxidative phosphorylation (electron transport)
pathway.
• The net result of these two closely linked pathways
is the oxidation of nutrients to produce usable
chemical energy in the form of ATP.
 THE CITRIC ACID CYCLE
• (Eukaryotes) Occurs in the matrix of the
mitochondrion.
• (Prokaryotic) the TCA reaction sequence is
performed in the cytosol.
 THE CITRIC ACID CYCLE
• The citric acid cycle itself was finally identified
in 1937 by Hans Adolf Krebs while at the
University of Sheffield, for which he received
the Nobel Prize for Physiology or Medicine in
1953.
 THE CITRIC ACID CYCLE
• The chemical reactions of the citric acid cycle
take place in the mitochondrial matrix where
the needed enzymes are found, except the
succinate dehydrogenase reaction that
involves FAD.
• The enzyme that catalyzes this reaction is an
integral part of the inner mitochondrial
membrane.
 THE CITRIC ACID CYCLE
• As one goes through the individual steps in
the process, two important types of reactions
may be observed:
1. Oxidation, which produces NADH and FADH2
2. Decarboxylation, wherein a carbon chain is
shortened by the removal of the carbon atom
as CO2
 THE CITRIC ACID CYCLE
THE STEPS
1. Formation of Citrate (CONDENSATION)
2. Formation of Isocitrate (ISOMERIZATION)
3. Oxidation of Isocitrate and formation of CO2
(OXIDATION AND DECARBOXYLATION).
4. Oxidation of α-ketoglutarate and formation
of CO2 (OXIDATION AND
DECARBOXYLATION).
 THE CITRIC ACID CYCLE
5. Thioester bond cleavage in Succinyl CoA and
Phosphorylation of GDP
(PHOSPHORYLATION).
6. Oxidation of Succinate (OXIDATION)
7. Hydration of Fumarate (HYDRATION)
8. Oxidation of L-Malate to regenerate
Oxaloacetate (OXIDATION).
SUMMARY
FADH2
FAD
 Reactions of Citric Acid Cycle
1. Citrate synthase: Formation of Citryl CoA intermediate.
 Reactions of Citric Acid Cycle
Binding of Oxaloacetate to the enzyme results in
conformational change which facilitates the binding of the
next substrate, the acetyl Coenzyme A. There is a further
conformational change which leads to formation of products.
This mechanism of reaction is referred as induced fit model.
• At cellular pH, citric acid is actually present as
citrate ion. Despite this, the name of the cycle
is citric acid cycle, which refers to the
molecular, rather than the ionic form of the
substance.
• A synthase is an enzyme that makes a new
covalent bond during a reaction without the
direct involvement of an ATP molecule.
 2. Aconitase:
• This enzyme catalyses the isomerization reaction by
removing and then adding back the water (H and OH ) to
cis-aconitate in at different positions.
• Isocitrate is consumed rapidly by the next step thus
deriving the reaction in forward direction.
3. Isocitrate dehydrogenase:
• There are two isoforms of this enzyme, one
uses NAD+ and other uses NADP+ as electron
acceptor.
BACK
• The key happenings in step 3 (Isocitrate
dehydrogenase) are the following:

1. The hydroxyl group of isocitrate is oxidized to

a ketone group.
2. NAD+ is converted to its reduced form,
NADH.
3. A carboxyl group from the original
oxaloacetate is removed as Carbon Dioxide.
 4. α-Ketoglutarate dehydrogenase:
• This is a complex of different enzymatic
activities similar to the pyruvate
dehydogenase complex.
• It has the same mechanism of reaction with
E1, E2 and E3 enzyme units. NAD+ is an
electron acceptor.
• The key happenings in step 4:
1. A second NAD+ is converted to its reduced form
NADH.
2. A second carboxyl group is removed as carbon
dioxide.
3. Coenzyme A reacts with the decarboxylation product
succinate to produce succinyl CoA, a compound with a
high energy thioester bond. This is the second
involvement of coenzyme A molecule in the cycle.
( The other CoA involvement is in step 1)
 5. Succinyl CoA synthetase:
• Succinyl CoA, like Acetyl CoA has a thioester
bond with very negative free energy of
hydrolysis.
• In this reaction, the hydrolysis of the thioester
bond leads to the formation of phosphoester
bond with inorganic phosphate.
Continuation: This phosphate is transferred
to Histidine residue of the enzyme and this
high energy, unstable phosphate is finally
transferred to GDP resulting in the
generation of GTP.
• The thioester bond in Succinyl CoA is
a strained bond.
• Its hydrolysis releases energy, which
is trapped by GTP formation.
• The function of the GTP produced is
similar to that of ATP: to store
energy in the form of a high energy
phosphate bond.
 6. Succinate Dehydrogenase:
• Oxidation of succinate to fumarate.
• This is the only citric acid cycle enzyme that is
tightly bound to the inner mitochondrial
membrane. It is an FAD dependent enzyme.
• Malonate has similar structure to Succinate, and
it competitively inhibits SDH.
BACK
FADH2

FAD
• Fumarate, with its trans double bond, is
an essential metabolic intermediate in
both plants and animals.
• Its isomer, with a cis-double bond, is
called maleate, and it is toxic and
irritating to tissues.
• Succinate dehydrogenase produces only
the trans isomer of this unsaturated
diacid.
 7. Fumarase:
• Hydration of Fumarate to malate: It is a
highly stereospecific enzyme.
• Cis-Maleate (the cis form of fumarate) is
not recognized by this enzyme.
 8. L-Malate dehydrogenase:
Oxidation of malate to oxaloacetate:
• It is an NAD+ dependent enzyme.
• Reaction is pulled in forward direction by the next
reaction (citrate synthase reaction) as the
oxaloacetate is depleted at a very fast rate.
SUMMARY OF THE CITRIC ACID CYCLE

Acetyl CoA + 3NAD+ + FAD + GDP

+ Pi + 2H20 ==
2 CO2 + CoA-SH + 3 NADH + 2H+
+ FADH2 + GTP

*products
 Important Cycle Features
1. The “fuel” for the cycle is Acetyl-CoA,
obtained from the breakdown of
carbohydrates, fats and proteins.
2. Four of the cycle reactions involve oxidation
and reduction. The oxidizing agent is either
NAD(3) or FAD(1). The operation of this cycle
depends on the availability of these oxidizing
agents.
3. In redox reactions, NAD+ is the oxidizing
agent when a CARBON-OXYGEN double bond
is formed. FAD is the oxidizing agent when a
CARBON-CARBON double bond is formed.
4. The three NADH and one FADH2 that are
formed during the cycle carry electrons and
H+ to the ETC through ATP is synthesized.
5. Two carbon atoms enter the cycle as the
acetyl unit of acetyl CoA, and two carbon
atoms leave the cycle as two molecules of
CO2.
• The carbon atoms that enter and leave are not the
same ones. The carbon atoms that leave during one
turn of the cycle are carbon atoms that entered
during the previous term of the cycle.
6. Four B vitamins are necessary for the proper
functioning of the cycle:
• riboflavin (in both FAD and the alpha ketoglutarate
dehydrogenase complex),
• nicotinamide in NAD,
• pantothenic acid in CoA-SH, and
• thiamine in alpha ketoglutarate dehydrogenase
complex.
7. One high-energy GTP molecule
is produced by phosphorylation
CAN CITRATE

I ISOCITRATE

KEEP on α-KETOGLUTARATE

SELLING SUCCINYL COA

SUBSTANCES SUCCINATE

FOR FUMARATE

MONEY, MALATE

OFFICER? OXALOACETATE
 SUE SYNTHASE CITRATE SYNTHASE

AND ACONITASE ACONITASE

DANNY DEHYDROGENASE ISOCITRATE

DEHYDROGENASE
DANCE DEHYDROGENASE α-KETOGLUTARATE
DEHYDROGENASE
SALSA, SYNTHETASE SUCCINYL COA SYNTHETASE

DISCO, DEHYDROGENASE SUCCINATE DEHYDROGENASE

FOXTRO FUMARASE FUMARASE

T, &
DAB DEHYDROGENASE MALATE DEHYDROGENASE
 Cheap CONDENSATION

Indians ISOMERATION

OrDer OXIDATION & DECARBOXYLATION

OrDinary OXIDATION & DECARBOXYLATION

Perfumes PHOSPHORYLATION

Only to OXIDATION

Hide their HYDRATION

Odor OXIDATION
 ELECTRON TRANSPORT CHAIN
• A series of biochemical reactions in which
electrons and hydrogen ions from NADH and
FADH2 are passed to intermediate carriers
and then ultimately react with molecular
oxygen to form water.
• NADH and FADH2 are oxidized in the process.
 ELECTRON TRANSPORT CHAIN
• Electrons passed in each step of ETC lose some
energy.
• Some of these lost energy are used to make ATP
from GDP.
• Inner mitochondial membrane- enzymes and
electron carriers are located.
 PROTEIN COMPLEXES:
• Bound to membrane
• COMPLEX I: NADH-coenzyme Q reductase
• COMPLEX II: Succinate-coenzyme Q reductase
• COMPLEX III: Coenzyme Q-cytochrome c reductase
• COMPLEX IV: Cytochrome c oxidase

• Coenzyme Q and Cytochrome-electron carriers

 COMPLEX I
• COMPLEX I: NADH-coenzyme Q reductase (NADH CoQ)
• LARGEST (40 subunits)
• FMN and Fe-S proteins (FESP)
STEPS:
1. Interaction of NADH with FMN.
• NAD is oxidized to NAD+, passes 2 H ions and e to
FMN, forming FMH2
2. Transfer of e from FMNH2 through Fe-S proteins.
• FMNH2 releases 2 H ions to the solution.
• This needs 2 Fe-SP molecules to accomodate the
release.
 COMPLEX I
STEPS:
3. Fe(II)SP is reconverted into Fe(III)SP, as each two
Fe(II)SP units passes an e to CoQ (CoQ to CoQH2).
• CoQ and CoQH2 are lipid soluble and can move
laterally within the mitochondrial membrane.
• CoQH2 shuttles e to COMPLEX III.
 COMPLEX III
• 11 subunits
• Contains FeSPs and Cytochromes
• Cytochrome: heme-containing protein in which
reversible oxidation and reduction of an iron atom
occur.
 COMPLEX III
STEPS:
1. Oxidation of CoQH2 to CoQ
• H+ produced go into cellular solution.
• Electron transport proceeds from CoQH2 to an
FeSP, to cyt b, then to another FeSP, to cyt c1, and
finally to cyt c.
• Cyt c can move laterally in the intermembrane
space, delivers its e to COMPLEX IV.
• Cyt c is the only water-soluble cytochrome.
 COMPLEX IV
• 13 subunits, including 2 cytochromes
• Cyt c to Cyt a, then cyt a3.
• Electrons from cyt a3 and H+ ions from cellular
solution combine with Oxygen to form water.
• 95% of the oxygen used by cells serve as the final
electron acceptor for the ETC.
 COMPLEX II
• 4 subunits, including 2 FESP molecules.
• Process FADH2
• CoQ is associated with the operations in COMPLEX
II in a manner similar to its actions in COMPLEX I.
• CoQ is the final recipient of the e from FADH2 (FeSP
as intermediaries).
• Complex I and II both produce CoQH2.