Lecture 1_Overview of Major Metabolic Pathways

FTAP6225
Advanced Human Nutrition

Lecture 1: Overview of major
metabolic pathways
Prof. Lifoter Kenneth Navti

Outline
 Introduction
 Glycolysis
 Gluconeogenesis
 Pentose phosphate pathway
 Kreb’s cycle
 Fatty acid catabolism
 Electron transfer chain
2
Introduction
 Metabolism can be defined as the sum of all the
enzyme-catalyzed reactions that take place in
cells.
 Metabolism is highly coordinated, purposeful
activity in which many sets of interrelated multi-
enzyme systems participate, exchanging
 both matter and energy between the cell and its
environment. Metabolism has four specific
functions:
3
Introduction
Functions of metabolism
 To obtain chemical energy from fuel molecules or
from absorbed sunlight.
 To convert exogenous nutrients into the building
blocks, or precursors, of macro-molecular cell
components.
 To assemble such building blocks into proteins,
nucleic acids, lipids and other cell components.
 To form and degrade bio-molecules required in
specialized functions of cells.
4
Introduction
Metabolism can be divided into 2 phases –
Catabolism and Anabolism.
 Catabolism is the degradative phase of
metabolism in which complex nutrient molecules
(lipids, carbohydrates and proteins) are broken
down to simpler end products such as C02, H20
and NH3 and is accompanied by the synthesis of
ATP.
5
Introduction
 Anabolism refers to biosynthetic processes in
which simple precursor molecules are
enzymatically converted into the molecular
components of cells, such as nucleic acids,
proteins, lipids and polysaccharides.
 Biosynthesis requires the input of ATP which is
provided by catabolism.
 There is also often a requirement for reducing
power in the form of NADPH.
6
Introduction
Stages of metabolism
 Stage 1: The nutrient macro-molecules are
broken down into their respective building blocks
– Proteins will yield amino acids, polysaccharides
give rise to carbohydrate units that are
convertible to glucose and lipids are broken down
into glycerol and fatty acids and other
components.
7
Introduction
 Stage 2: the many different products of stage 1
are collected and converted/degraded again into
simpler metabolic intermediates. Thus, hexoses,
pentoses and glycerol are degraded via the 3-
carbon intermediate pynuvic acid to yield a single
carbon species, acetyl CoA.
 The various fatty acids and amino acids are
broken down to form acetyl CoA and a few other
end products.
8
Introduction
 Stage 3: The combustion of the acetyl groups of
acetyl CoA by the nitric acid cycles and oxidative
phosphorylation to produce C02 and H20
represent stage 3 of catabolism. C02 and H20 are
the ultimate waste products of aerobic
catabolism.
9
Introduction
10
Glycolysis
11
Outline
 Introduction
 General information on carbohydrate metabolism
 Glycolysis
 The glycolytic pathway
 Fates of pyruvate
12
Introduction
 Greek word- glycos meaning sweet/sugar and lysis
meaning dissolution
 Glycolysis is the first step in the breakdown of
glucose to extract energy for cellular metabolism.
Glycolysis consists of an energy-requiring phase
followed by an energy-releasing phase.
 Glycolysis is a series of reactions that extract energy
from glucose by splitting it into two three-carbon
molecules called pyruvates.
 Sequence of reactions converting glucose/glycogen
to pyruvate/lactate with production of ATP.
13
Introduction
 It is also known as the Embden-Meyerhof-Parnas
pathway.
 Glycolysis, occurs, at least in part, in almost every
living cell.
 This series of reactions is believed to be among the
oldest of all the biochemical pathways.
 Both the enzymes and the number and mechanisms
of the steps in the pathway are highly conserved in
prokaryotes and eukaryotes.
14
Introduction
 In organisms that perform cellular respiration,
glycolysis is the first stage of this process. However,
glycolysis doesn’t require oxygen, and many
anaerobic organisms—organisms that do not use
oxygen—also have this pathway.
 Reversal of glycolysis along with the alternate
arrangement at the irreversible steps will result in the
synthesis of glucose (Gluconeogenesis)
15
Carbohydrate metabolism
 Major pathways in carbohydrate metabolism
16
 In animals, excess glucose is converted to its storage
form, glycogen, by glycogenesis. When glucose is
needed as a source of energy or as a precursor
molecule in biosynthetic processes, glycogen is
degraded by glycogenolysis.
 Glucose can be converted to ribose-5-phosphate (a
component of nucleotides) and NADPH (a powerful
reducing agent) by means of the pentose phosphate
pathway.
17
 Glucose is oxidized by glycolysis, an energy-
generating pathway that converts it to pyruvate. In
the absence of oxygen, pyruvate is converted to
lactate.
 When oxygen is present, pyruvate is further
degraded to form acetyl-CoA. Significant amounts of
energy in the form of ATP can be extracted from
acetyl-CoA by the citric acid cycle and the electron
transport system.
18
 Note that carbohydrate metabolism is inextricably
linked to the metabolism of other nutrients.
 For example, acetyl-CoA is also generated from the
breakdown of fatty acids and certain amino acids.
 When acetyl-CoA is present in excess, a different
pathway converts it into fatty acids.
19
Glycolysis
Glycolysis, which consists of 10 reactions, occurs in two
stages:
 1. Glucose is phosphorylated twice and cleaved to
form two molecules of glyceraldehyde-3-phosphate
(G-3-P). The two ATP molecules consumed during
this stage are like an investment, because this stage
creates the actual substrates for oxidation in a form
that is trapped inside the cell.
20
Glycolysis
 2. Glyceraldehyde-3-phosphate is converted to
pyruvate. Four ATP and two NADH molecules are
produced. Because two ATP were consumed in
stage 1, the net production of ATP per glucose
molecule is 2.
21
Glycolysis
 The glycolytic pathway can be summed up in the
following equation:
22
The glycolytic pathway
23
The glycolytic pathway: Rxn 1
1. Synthesis of glucose-6-phosphate
24
 Immediately after entering a cell, glucose and other
sugar molecules are phosphorylated.
 Phosphorylation prevents transport of glucose out of
the cell and increases the reactivity of the oxygen in
the resulting phosphate ester.
 Several enzymes, called the hexokinases, catalyze
the phosphorylation of hexoses in all cells in the
body.
25
 ATP, a cosubstrate in the reaction, is complexed with
Mg2+. (ATP-Mg2+ complexes are common in kinase-
catalyzed reactions).
 Under intracellular conditions the reaction is
irreversible; that is, the enzyme has no ability to retain
or accommodate the product of the reaction in its
active site, regardless of the concentration of G-6-P.
26
2. Conversion of glucose-6-phosphate to fructose-6-
phosphate
27
 During reaction 2 of glycolysis, the open chain form
of the aldose glucose-6-phosphate is converted to
the open chain form of the ketose fructose-6-
phosphate by phosphoglucose isomerase (PGI) in a
readily reversible reaction.
 Recall that the isomerization reaction of glucose and
fructose involves an enediol intermediate. This
transformation makes C-1 of the fructose product
available for phosphorylation. The hemiacetal
hydroxy group of glucose-6-phosphate is more
difficult to phosphorylate.
28
3. The phosphorylation of fructose-6-phosphate
29
 Phosphofructokinase-1 (PFK-1) irreversibly catalyzes
the phosphorylation of fructose-6-phosphate to form
fructose-1,6-bisphosphate.
 The PFK-1-catalyzed reaction is irreversible under
cellular conditions. It is, therefore, the first committed
step in glycolysis. Unlike glucose-6-phosphate and
fructose-6-phosphate, the substrate and product,
respectively, of the previous reaction, fructose-1,6-
bisphosphate cannot be diverted into other pathways.
Investing a second molecule of ATP serves several
purposes.
30
 First of all, because ATP is used as the
phosphorylating agent, the reaction proceeds with a
large decrease in free energy. After fructose-1,6-
bisphosphate has been synthesized, the cell is
committed to glycolysis.
 Because fructose-1,6-bisphosphate eventually splits
into two trioses, another purpose for phosphorylation
is to prevent any later product from diffusing out of
the cell because charged molecules cannot easily
cross membranes.
31
4. Cleavage of fructose-1,6-bisphosphate
32
 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. Aldol cleavages are the
reverse of aldol condensations. In aldol cleavages an
aldehyde and a ketone are products.
33
5. The interconversion of glyceraldehyde-3-phosphate
and dihydroxy acetone phosphate
34
 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.
 After this reaction, the original molecule of glucose
has been converted to two molecules of G-3-P.
35
6. Oxidation of glyceraldehye-3-phosphate
36
 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, a
tetramer composed of four identical subunits. Each
subunit contains one binding site for G-3-P and
another for NAD+, an oxidizing agent.
37
 As the enzyme forms a covalent thioester bond with
the substrate a hydride ion (H-) is transferred to NAD+
in the active site.
 NADH, the reduced form of NAD+, then leaves the
active site and is replaced by an incoming NAD+. The
acyl enzyme adduct is attacked by inorganic
 phosphate and the product leaves the active site.
38
7. Phosphoryl group transfer
39
 In this reaction ATP is synthesized as
phosphoglycerate kinase catalyzes the transfer of the
high-energy phosphoryl group of glycerate-1,3-
bisphosphate to ADP.
 Reaction 7 is an example of a substrate-level
phosphorylation. Because the 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)
40
 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.
ATP synthesis later in the pathway represents a net
gain.
41
8. The interconversion of 3-phosphoglycerate and 2-
phosphoglycerate
42
 Glycerate-3-phosphate has a low phosphoryl group
transfer potential. As such, it is a poor candidate for
further ATP synthesis.
 Cells convert glycerate-3-phosphate with its energy-
poor phosphate ester to phosphoenolpyruvate (PEP),
which has an exceptionally high phosphoryl group
transfer potential
43
 In the first step in this conversion (reaction 8),
phosphoglycerate mutase catalyzes the conversion
of a C-3 phosphorylated compound to a C-2
phosphorylated compound through a two-step
addition/elimination cycle.
44
9. Dehydration of 2-phosphoglycerate
45
 Enolase catalyzes the dehydration of glycerate-2-
phosphate to form PEP.
 PEP has a higher phosphoryl group transfer potential
than does glycerate-2-phosphate because it contains
an enol-phosphate group instead of a simple
phosphate ester. The reason for this difference is
made apparent in the next reaction.
46
 Aldehydes and ketones have two isomeric forms. The
enol form contains a carbon-carbon double bond and
a hydroxyl group. Enols exist in equilibrium with the
more stable carbonyl-containing keto form.
 The interconversion of keto and enol forms, also
called tautomers, is referred to as tautomerization.
 This tautomerization is restricted by the presence of
the phosphate group, as is the resonance
stabilization of the free phosphate ion. As a result,
phosphoryl transfer to ADP in reaction 10 is highly
favored.
47
10. Synthesis of pyruvate
48
 In the final reaction of glycolysis, pyruvate kinase
catalyzes the transfer of a phosphoryl group from
PEP to ADP. Two molecules of ATP are formed for
each molecule of glucose.
49
The glycolytic pathway
50
The fates of pyruvate
 In terms of energy, the result of glycolysis is the
production of two ATPs and two NADHs per molecule
of glucose. Pyruvate, the other product of glycolysis,
is still an energy-rich molecule, which can yield a
substantial amount of ATP.
 Whether or not further energy can be produced,
however, depends on the cell type and the availability
of oxygen.
51
 Under aerobic conditions, most cells in the body
convert pyruvate into acetyl-CoA, the entry-level
substrate for the citric acid cycle, an amphibolic
pathway that completely oxidizes the two acetyl
carbons to form CO2 and and the reduced molecules
NADH and FADH2. (An amphibolic pathway functions
in both anabolic and catabolic processes).
52
 The electron transport system, a series of oxidation-
reduction reactions, transfers electrons from NADH
and FADH2 to O2 to form water. The energy that is
released during electron transport is coupled to a
mechanism that synthesizes ATP.
 Under anaerobic conditions, further oxidation of
pyruvate is impeded. A number of cells and
organisms compensate by converting this molecule
to a more reduced organic compound and
regenerating the NAD+ required for glycolysis to
continue.
53
 This process of NAD+ regeneration is referred to as
fermentation. Muscle cells, red blood cells, and
certain bacterial species (e.g., Lactobacillus) produce
NAD+ by transforming pyruvate into lactate.
54
 In rapidly contracting muscle cells, the demand for
energy is high. After the O2 supply is depleted, lactic
acid fermentation provides sufficient NAD+ to allow
glycolysis (with its low level of ATP production) to
continue for a short time.
55
56
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 some cells
(e.g., yeast), ethanol and CO2 are produced (middle).
 In others (e.g., muscle cells), homolactic fermentation
occurs in which lactate is the only organic product
(right). Some microorganisms use heterolactic
fermentation reactions (not shown) that produce
other acids or alcohols in addition to lactate.
 In all fermentation processes, the principal purpose is
to regenerate NAD+ so that glycolysis can continue.
57
Gluconeogenesis
58
Introduction
 Gluconeogenesis, the formation of new glucose
molecules from non-carbohydrate precursors, occurs
primarily in the liver. Precursor molecules include
lactate, pyruvate, glycerol, and certain -keto acids
(molecules derived from amino acids).
 Under certain conditions (i.e., metabolic acidosis or
starvation) the kidney can make small amounts of
new glucose. Between meals adequate blood
glucose levels are maintained by the hydrolysis of
liver glycogen.
59
Introduction
 When liver glycogen is depleted (e.g., owing to
prolonged fasting or vigorous exercise), the
gluconeogenesis pathway provides the body with
adequate glucose.
 Brain and red blood cells rely exclusively on glucose
as their energy source.
60
Gluconeogenesis reactions
 The reaction sequence in gluconeogenesis is largely
the reverse of glycolysis.
 Recall, however, that three glycolytic reactions (the
reactions catalyzed by hexokinase, PFK-1, and
pyruvate kinase) are irreversible.
 In gluconeogenesis, alternate reactions catalyzed by
different enzymes are used to bypass these
obstacles.
 The entire gluconeogenic pathway and its
relationship to glycolysis are illustrated in the next
slide.
61
Gluconeogenesis reactions
62
#1
1. 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):
63
#1
64
#1
 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.
 OAA is then decarboxylated and phosphorylated by
PEP car boxykinase in a reaction driven by the
hydrolysis of guanosine triphosphate (GTP):
65
#1
66
#1
 PEP carboxykinase is found within the mitochondria
of some species and in the cytoplasm of others. In
humans this enzymatic activity is found in both
compartments.
 Because the inner mitochondrial membrane is
impermeable to OAA, cells that lack mitochondrial
PEP carboxykinase transfer OAA into the cytoplasm
by using the malate shuttle.
 In this process, OAA is converted into malate by
mitochondrial malate dehydrogenase.
67
#1
68
#1
 After the transport of malate across mitochondrial
membrane, the reverse reaction (to form OAA and
NADH) is catalyzed by cytoplasmic malate
dehydrogenase.
 The malate shuttle allows gluconeogenesis to
continue because it provides the NADH required for
the reaction catalyzed by glyceraldehyde-3-
phosphate dehydrogenase.
69
#2
2. 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:
 Fructose-1,6-bisphosphatase is an allosteric enzyme.
 Its activity is stimulated by citrate and inhibited by
AMP and fructose-2,6-bisphosphate.
70
#2
71
#3
3. 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.
72
#3
 Each of the foregoing reactions is matched by an
opposing irreversible reaction in glycolysis. Each set
of such paired reactions is referred to as a substrate
cycle.
 Because they are coordinately regulated (an activator
of the enzyme catalyzing the forward reaction serves
as an inhibitor of the enzyme catalyzing the reverse
reaction), very little energy is wasted, even though
both enzymes may be operating at some level at the
same time.
73
#3
 Flux control (regulation of the flow of substrate and
removal of product) is more effective if transient
accumulation of product is funneled back through the
cycle.
 The catalytic velocity of the forward enzyme will
remain high if the concentration of the substrate is
maximized. The gain in catalytic efficiency more than
makes up for the small energy loss in recycling the
product.
74
#3
 Gluconeogenesis is an energy-consuming process.
Instead of generating ATP (as in glycolysis),
gluconeogenesis requires the hydrolysis of six high
energy phosphate bonds.
75
Gluconeogenesis regulation
 As with other metabolic pathways, the rate of
gluconeogenesis is affected primarily by substrate
availability, allosteric effectors, and hormones. Not
surprisingly, gluconeogenesis is stimulated by high
concentrations of lactate, glycerol, and amino acids.
 A high-fat diet, starvation, and prolonged fasting
make large quantities of these molecules available.
76
Gluconeogenesis regulation
 The four key enzymes in gluconeogenesis (pyruvate
carboxylase, PEP carboxykinase, fructose-1,6-
bisphosphatase, and glucose-6-phosphatase) are
affected to varying degrees by allosteric modulators.
For example, fructose-1,6-bisphosphatase is
activated by citrate and inhibited by AMP and
fructose-2,6-bisphosphate.
 Acetyl-CoA activates pyruvate carboxylase.
77
Allosteric regulation of glycolysis
and gluconeogenesis
 As with other biochemical pathways, hormones affect
gluconeogenesis by altering the concentrations of
allosteric effectors and the key rate-determining
enzymes. As mentioned previously, glucagon
depresses the synthesis of fructose-2,6-
bisphosphate, which releases the inhibition of
fructose-1,6-bisphosphatase, and inactivates the
glycolytic enzyme pyruvate kinase.
 Hormones also influence gluconeogenesis by altering
enzyme synthesis.
78
and gluconeogenesis
 For example, the synthesis of gluconeogenic
enzymes is stimulated by cortisol, a steroid hormone
produced in the cortex of the adrenal gland that
facilitates the body’s adaptation to stressful
situations.
 Finally, insulin action leads to the synthesis of new
molecules of glucokinase, PFK-1 (SREBP1c-
induced), and PFK-2 (glycolysis favored). Insulin also
depresses the synthesis (also via SREBP1c) of PEP
carboxykinase, fructose-1,6-bisphosphatase, and
glucose-6-phosphatase.
79
and gluconeogenesis
 The hormones that regulate glycolysis and
gluconeogenesis alter the phosphorylation state of
certain target proteins in the liver cell, which in turn
modifies gene expression.
 The key point to remember is that insulin and
glucagon have opposing effects on carbohydrate
metabolism.
80
and gluconeogenesis
 The direction of metabolite flux, (i.e., whether either
glycolysis or gluconeogenesis is active) is largely
determined by the ratio of insulin to glucagon. After a
carbohydrate meal, the insulin/glucagon ratio is high
and glycolysis in the liver predominates over
gluconeogenesis.
 After a period of fasting or following a high-fat, low-
carbohydrate meal, the insulin/glucagon ratio is low
and gluconeogenesis in the liver predominates over
 glycolysis.
81
and gluconeogenesis
 The availability of ATP is the second important
regulator in the reciprocal control of glycolysis and
gluconeogenesis in that high levels of AMP, the low-
energy hydrolysis product of ATP, increase the flux
through glycolysis at the expense of
gluconeogenesis, and low levels of AMP increase the
flux through gluconeogenesis at the expense of
glycolysis.
82
Pentose phosphate pathway
83
Introduction
 The pentose phosphate pathway (PPP) is an
alternative metabolic pathway for glucose
oxidation in which no ATP is generated.
 Its principal products are NADPH, a reducing
agent required in several anabolic processes,
and ribose-5-phosphate,a structural component
of nucleotides and nucleic acids.
 The pentose phosphate pathway occurs in the
cytoplasm in two phases: oxidative and
nonoxidative.
84
Introduction
 In the oxidative phase of the pathway, the
conversion of glucose-6-phosphate to ribulose-5-
phosphate is accompanied by the production of
two molecules of NADPH.
 The nonoxidative phase involves the
isomerization and condensation of a number of
different sugar molecules.
 Three intermediates in this process that are
useful in other pathways are ribose-5-phosphate,
fructose-6-phosphate, and glyceraldehyde-3-
phosphate.
85
Oxidative phase of PPP
 The oxidative phase of the PPP consists of three
reactions.
 In the first reaction, glucose-6-phosphate
dehydrogenase (G-6-PD) catalyzes the oxidation of
glucose-6-phosphate. 6-Phosphogluconolactone
and NADPH are products in this reaction.
 6-Phospho-D-glucono-δ-lactone is then hydrolyzed
to produce 6-phospho-D-gluconate. A second
molecule of NADPH is produced during the
oxidative decarboxylation of 6-phosphogluconate, a
reaction that yields ribulose-5-phosphate
86
 A substantial amount of the NADPH required for
reductive processes (i.e., lipid biosynthesis) is
supplied by these reactions.
 For this reason this pathway is most active in
cells in which relatively large amounts of lipids
are synthesized, (e.g., adipose tissue, adrenal
cortex, mammary glands, and the liver)
87
88
Non-oxidative phase of PPP
 The nonoxidative phase of the pathway begins
with the conversion of ribulose-5-phosphate to
ribose-5-phosphate by ribulose-5-phosphate
isomerase or to xylulose-5-phosphate by
ribulose-5-phosphate epimerase.
 During the remaining reactions of the pathway
transketolase and transaldolase catalyze the
interconversions of trioses, pentoses, and
hexoses.
89
 Transketolase catalyzes two reactions. In the first
reaction, the enzyme transfers a two-carbon unit
from xylulose-5-phosphate to ribose-5-
phosphate, yielding glyceraldehyde-3-phosphate
and sedoheptulose-7-phosphate.
 In the second transketolase-catalyzed reaction, a
two-carbon unit from another xylulose-5-
phosphate molecule is transferred to erythrose-4-
phosphate to form a second molecule of
glyceraldehyde-3-phosphate and fructose-6-
phosphate
90
Non-oxidative phase
 Transaldolase transfers three-carbon units from a
ketose to an aldose.
 In the reaction catalyzed by transaldolase, a
three- carbon unit is transferred from
sedoheptulose-7-phosphate to glyceraldehyde-3-
phosphate. The products formed are fructose-6-
phosphate and erythrose-4-phosphate.
91
 The result of the nonoxidative phase of the
pathway is the synthesis of ribose-5-phosphate
and the glycolytic intermediates glyceraldehyde-
3- phosphate and fructose-6-phosphate.
92
93
 When pentose sugars are not required for
biosynthetic reactions, the metabolites in the
nonoxidative portion of the pathway are
converted into glycolytic intermediates that can
then be further degraded to generate energy or
converted into precursor molecules for
biosynthetic processes
94
Glycolysis and PPP
95
Regulation of PPP
 The pentose phosphate pathway is regulated to
meet the cell’s moment by-moment requirements
for NADPH and ribose-5-phosphate.
 The oxidative phase is very active in cells such
as red blood cells or hepatocytes in which
demand for NADPH is high.
 In contrast, the oxidative phase is virtually absent
in cells (e.g., muscle cells) that synthesize little or
no lipid
96
Regulation of PPP
 G-6-PD catalyzes a key regulatory step in the
pentose phosphate pathway. Its activity is
inhibited by NADPH and stimulated by GSSG,
the oxidized form of glutathione, an important
cellular antioxidant and glucose-6-phosphate.
 In addition, diets high in carbohydrate increase
the synthesis of both G-6-PD and
phosphogluconate dehydrogenase.
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Citric acid cycle
98
Role
 The citric acid cycle, also known as the TCA
(tricarboxylic acid) cycle or Krebs cycle (after its
discoverer in 1937), is used to oxidize the pyruvate
formed during the glycolytic breakdown of glucose
into CO2 and H2O.
 It also oxidizes acetyl CoA arising from fatty acid
degradation, and amino acid degradation products.
 In addition, the cycle provides precursors for many
biosynthetic pathways.
99
Location
 The citric acid cycle operates in the mitochondria of
eukaryotes and in the cytosol of prokaryotes.
Succinate dehydrogenase, the only membrane-
bound enzyme in the citric acid cycle, is embedded in
the inner mitochondrial membrane in eukaryotes and
in the plasma membrane in prokaryotes.
100
The cycle
 The cycle forms the central part of a three-step
process which oxidizes organic fuel molecules into
CO2 with the concomitant production of ATP.
101
The CAC cycle
102
Step 1: Oxidation of fuel
molecules to acetyl CoA
 A major source of energy is glucose which is
converted by glycolysis into pyruvate.
 Pyruvate dehydrogenase (a complex of three
enzymes and five coenzymes) then oxidizes the
pyruvate (using NAD+ which is reduced to NADH) to
form acetyl CoA and CO2.
 Since the reaction involves both an oxidation and a
loss of CO2, the process is called oxidative
decarboxylation.
103
Step 2: The citric acid cycle
 The cycle carries out the oxidation of acetyl groups
from acetyl CoA to CO2 with the production of four
pairs of electrons, stored initially in the reduced
electron carriers NADH and FADH2.
 The cycle has eight stages:
104
Eight stages
 Citrate (6C) is formed from the irreversible condensation of
acetyl CoA (2C) and oxaloacetate (4C) – catalyzed by citrate
synthase.
 Citrate is converted to isocitrate (6C) by an isomerization
catalyzed by aconitase. This is actually a two-step reaction
during which cis-aconitate is formed as an intermediate. It is the
cis-aconitate which gives the enzyme its name.
 Isocitrate is oxidized to α-ketoglutarate (5C) and CO2 by
isocitrate dehydrogenase. This mitochondrial enzyme requires
NAD+, which is reduced to NADH.
 4. α-Ketoglutarate is oxidized to succinyl CoA (4C) and CO2 by
the α–ketoglutarate dehydrogenase complex. Like pyruvate
dehydrogenase, this is a complex of three enzymes and uses
NAD+ as a cofactor.
105
Eight stages cont.
 Succinyl CoA is converted to succinate (4C) by succinyl CoA
synthetase. The reaction uses the energy released by cleavage
of the succinyl–CoA bond to synthesize either GTP (mainly in
animals) or ATP (exclusively in plants) from Pi and, respectively,
GDP or ADP.
 Succinate is oxidized to fumarate (4C) by succinate
dehydrogenase. FAD is tightly bound to the enzyme and is
reduced to produce FADH2.
 Fumarate is converted to malate (4C) by fumarase; this is a
hydration reaction requiring the addition of a water molecule.
 Malate is oxidized to oxaloacetate (4C) by malate
dehydrogenase. NAD+ is again required by the enzyme as a
cofactor to accept the free pair of electrons and produce NADH.
106
Step 3:
Oxidation of NADH and FADH2
 The NADH and FADH2 produced by the citric
acid cycle are reoxidized and the energy
released is used to synthesize ATP by oxidative
phosphorylation
107
Energy yield
 Each of the three NADH molecules produced per turn
of the cycle yields 3 ATPs and the single FADH2
yields 2 ATPs by oxidative phosphorylation
 One GTP (or ATP) is synthesized directly during the
conversion of succinyl CoA to succinate.
 Thus the oxidation of a single molecule of glucose via
the citric acid cycle produces 12 ATP molecules.
108
Regulation
 Regulation of the cycle is governed by substrate
availability, inhibition by accumulating products, and
allosteric feedback inhibition by subsequent
intermediates in the cycle.
 Three enzymes in the cycle itself are regulated
(citrate synthase, isocitrate dehydrogenase and -
ketoglutarate dehydrogenase) and so is the enzyme
which converts pyruvate to acetyl CoA to enter the
cycle, namely pyruvate dehydrogenase.
109
Regulation
 Citrate synthase is inhibited by citrate and also by
ATP
 Isocitrate dehydrogenase is inhibited by NADH and
ATP but activated by ADP;
 α-ketoglutarate dehydrogenase is inhibited by NADH
and succinyl CoA;
 Pyruvate dehydrogenase is inhibited by NADH and
acetyl CoA
110
Regulatory points of the CAC
111
Electron transport chain
112
Outline
 Electron transport chain (ETC) or respiratory
chain
 Mitochondrial organization
 Structural organization of ETC
 Major electron carriers
 Redox systems of the ETC
113
What is the electron transport
chain (ETC) or respiratory chain
 This is the final common pathway in aerobic cells
by which electrons derived from various
substrates are transferred to oxygen.
 ETC is series of highly organized oxidation-
reduction reactions.
114
Electron transport chain (ETC)
 ETC is the 4th and final stage of aerobic
respiration.
 Through ETC, the energy needed for the cellular
activities is released in the form of ATP.
 ETC is an O2 dependent process which occurs in
the inner mitochondrial membrane.
115
 The energy rich carbohydrates (Glu), fatty acids
and amino acids undergo a series of metabolic
reactions and finally get oxidized to CO2 and
H2O.
 The reducing equivalents from various metabolic
intermediates are transferred to NAD+ and FAD
to produce NADH and FADH2.
 The latter two reduced coenzymes pass through
the ETC or respiratory chain and finally reduce
O2 to H2O.
116
 The passage of electrons through the ETC is
associated with the loss of free energy.
 A part of this free energy is utilized to generate
ATP from ADP and Pi.…
117
Location of ETC
 ETC is localized in Mitochondria.
 Mitochondria are the centers for metabolic
oxidative reactions to generate reduced
coenzymes (NADH and FADH2) which in turn,
are utilized in ETC to liberate energy in the form
of ATP.
 Hence, the mitochondria is regarded as Power
House of the Cell.
118
Mitochondrial organization
5 distinct parts.
 the outer membrane
 the inner membrane
 the inter membrane space
 the cristae
 the matrix
119
Inner mitochondrial membrane
(IMM)
 ETC and ATP synthesizing system are located
on IMM.
 IMM is rich in proteins.
 It is impermeable to ions (H+, K+, Na+) and small
molecules (ADP, ATP).
 IMM is highly folded to form cristae.
 The surface area of the IMM is greatly increased
due to cristae.
120
Inner mitochondrial membrane
(IMM)
 The IMM possesses specialized particles (that
look like lollipops), the phosphorylating subunits
which are the centers for ATP production.
121
Structural organization of the ETC
122
123
 The IMM includes 5 distinct enzyme complexes,
denoted as Complex I, II, III, IV and V
 The complex I-IV are carriers of electrons while V
is responsible for ATP synthesis.
 Besides these enzyme complexes, there are
certain mobile electron carriers in ETC.
 These include NADH, Coenzyme Q, Cytochrome
C and Oxygen.
124
 The complexes (I-IV) and the mobile carriers are
collectively involved in the transport of electrons,
which ultimately combine with O2 to produce
H2O.
 Most of the O2 supplied to the body is utilized by
mitochondria for ETC.
125
Major electron carriers
Introduction:
 During the oxidation of food fuel molecules such
as glucose and fatty acids to produce energy, the
transfer of electrons (or protons) from the
electron donor molecule to the final electron
acceptor molecule is ensured by special electron
carriers, which are principally pyrimidine
nucleotides or flavins
126
Major electron carriers
 These carriers are sequentially arranged and are
responsible for the transfer of electrons from a
given substrate to ultimately combine with proton
and O2 to form H2O.
127
Nicotinamide nucleotides
Oxidized form Reduced form
128
Nicotinamide nucleotide
 Of the 2 coenzymes NAD+ and NADP+ derived
from the vit. Niacin, NAD+ is more actively
involved in the electron transport chain (ETC).
 NAD+ is reduced to NADH + H+ by
dehydrogenases with the removal of 2H atoms
from the substrate.
 The substrates include Gly-3-P, Pyruvate,
isocitrate, α-ketoglutarate, and malate.
129
Nicotinamide nucleotide
 NADPH + H+ produced by NADP+- dependent
dehydrogenase is not usually a substrate for
ETC.
 NADPH is more effectively utilized for anabolic
reactions
 Eg: Fatty acid synthesis, Cholesterol synthesis.
130
Flavoproteins
 Flavin mononucleotide (FMN)
131
Flavoproteins
 Flavine adenine dinucleotide (FAD)
132
Flavoproteins
 The enzyme NADH dehydrogenase (NADH-CoQ
reductase) is a flavo protein.
 FMN is the prosthetic group.
 FMN accepts 2 electrons and a proton to form
FMNH2
 NADH dehydrogenase is a complex enzyme
closely associated with non-heme iron proteins
(NHI) or iron-sulfur proteins.
 NADH+ H+ + FMN NAD+ + FMNH2
133
Flavoproteins
 SDH (Succinate-Co.Q reductase) is an enzyme
found in the inner mitochondrial membrane.
 It is also a flavoprotein with FAD as the
coenzyme.
 SDH can accept 2 H atoms(2H+ + 2e- ) from
succinate.
 Succinate + FAD Fumarate + FADH2
134
Coenzyme Q or ubiquinone
135
 Also called Ubiquinone since it is ubiquitous in
living system.
 It is a quinone derivative with a variable
isoprenoid side chain.
 The mammalian tissues possess a quinone with
10 isoprenoid units which is known as coenzyme
Q10 (CoQ10)
136
 CoQ is a lipophilic electron carrier.
 CoQ is not found in mitochondria
 Vit k performs similar function as CoQ
137
Cytochromes
138
Cytochromes
 Cytochromes are conjugated proteins.
 Contains heme group.
 The heme group of cyt differs from that of
Haemoglobin (Hb) and Myoglobin (Mb).
 The Iron of Heme in cyt is alternately oxidized
(Fe3+) and reduced (Fe2+), which is essential for
the transport of electrons in ETC.
 This is in contrast to the Hb and Mb iron which
remains in the Fe2+ state.
139
Cytochromes
 Cyt are identified by their characteristic
absorption spectra.
 Ferricytochromes show diffuse and non-
characteristic absorption spectra.
 Ferrocytochromes exhibit characteristic
absorption bands called α, β and γ–soret bands.
 Cytochromes are characterized into different
groups according to the light wavelength at which
the alpha band shows its peak(α-abs.max.)
140
Types of cytochromes
 Cyt. c:- Since it is largely available, it is the best
studied of the cytochromes.
 It is a central member of ETC with an
intermediate redox potential)
 Water soluble-loosely bound to inner
mitochondrial membrane, easy to extract.
 The iron content of cyt. c. is 0.38%
 Heme is attached with protein by means of 2
thioester linkages involving sulfur of 2 cystein
and apoprotein.
141
 Cyt. c is incapable of combining with O2/CO.
 A protein with 1-PPchain 104aa (mw12400-
13000)
 NADPH-Cyt.c.reductase can readily reduce Cyt.c
142
 Cyt. c1: Like cyt. C, it contains an
ironprotoporphyrinIX complex-heme-c.
 Incapable of combining with O2,CO,CN
143
 Cyt. b: Also a protoporphyrinIXcomplex-(heme-
b). But the apoprotein is different.
 Tightly bound to flavo proteins and ubiquinones
in the mitochondria
 It is thermostable and not easily extractable.
 It also does not react with O2,CO or CN-
 Normally its oxidation requires the presence of
Cyt. c, a, and a3.
144
Cytochrome oxidase (cyt a & a3)
 Constitute complex IV of the ETC.
 Both contain an identical type of iron porphyrin
complex
 Inspite of identical hemes, cyt. a & a3 differ in
electron affinity and bio.activity. This is because
of their location of hemes
 One heme is located along with one Cu ion. This
heme is called heme-a
 This Cyt. a functions as anaerobic oxidizing unit.
145
Cytochrome oxidase (cyt a & a3)
 The other heme is located along with the 2nd Cu
ion and is called heme-a3 (functions as aerobic
reducing unit).
 Cyt. a does not react with O2,CO/CN whereas
Cyt.a3 is autooxidizable and forms compounds
with CO & CN-.
146
Iron-sulphur proteins
147
 A group of quinones has been found to be
present in mitochondria namely FeS, Fe2S2,
Fe4S4 and Fe3S4 etc.,
 FeS proteins exist in the oxidized (Fe3+) or
reduced (Fe2+).
 About 6 FeS proteins connected with ETC have
been identified.
 The mechanism of FeS proteins in ETC is not
clearly understood.
148
 One FeS participates in the transfer of electrons
from FMN to Co.Q
 Other FeS proteins associated with cyt. b and
cyt. c1 participate in the transport of electrons.
 Vit E, D and plastoquinones also involved in
ETC.
149
 FeS: It has a single Fe coordinated to the side
chain-SH groups of 4 cystein residues.
 Fe2S2: It contains 2 iron atoms, 2 inorganic
sulfides and 4 –SH groups.
 Each iron is linked to 2-SH and 2-sulfur groups.
 Fe4S4: It consists of 4 iron atoms and 4 cys-SH
groups and 4 inorganic sulfides. Each iron
remains linked to 1-SH, 3-inorganic sulfides while
each sulfide is coordinated to 3 iron atoms.
150
 Fe3S4: It consists of 3 Fe, 4 -SH and 4 inorganic
sulfides.
 Each FeS protein transfers only one electron at a
time.
 The enzymes may have one or more of the
combinations
151
Redox systems of the ETC
152

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FTAP6225

Advanced Human Nutrition

Prof. Lifoter Kenneth Navti

Oxidized form Reduced form

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