Lipid Metabolism

Dr Hasan Akbar Khan

• Lipids play a variety of cellular roles, some only recently
recognized
1. They are the principal form of stored energy in most organisms.
2. Major constituents of cellular membranes.
3. Specialized lipids serve as
i. Pigments (Retinal, Carotene)
ii. Cofactors (Vitamin K)
iii. Detergents (Bile Salts)
iv. Hormones (Vitamin D Derivatives, Sex Hormones)
v. Extracellular and intracellular messengers (eicosanoids,
phosphatidylinositol derivatives)
vi. Anchors for membrane proteins (covalently attached fatty
acids, prenyl groups, and phosphatidylinositol)
2
• Like most other biosynthetic pathways, the
biosynthetic pathways for some of the most
common cellular lipids are endergonic and
reductive

• They use ATP as a source of metabolic energy and

a reduced electron carrier (usually NADPH) as a
reductant

3
FATTY ACID SYNTHESIS (Lynen’s
Spiral)

• Fatty acids are synthesized whenever an

excess of calories is ingested

• The major source of carbon for the

synthesis of fatty acids are dietary
carbohydrates
4
• When an excess of dietary carbohydrates are
consumed, glucose is converted to acetyl CoA,
which provides the 2-carbon units that condense
in a series of reactions on the fatty acid synthase
complex, producing palmitate (16 C)

• Palmitate is then converted to other fatty acids

5
• The fatty acid synthase

complex is located in the

cytosol, and, therefore, it uses

Cytosolic Acetyl-CoA

6
The Main Pathway for De Novo Synthesis of
Fatty Acids (Lipogenesis) Occurs in the Cytosol

• This system is present in many tissues, including

Liver, Kidney, Brain, Lungs, Mammary Glands,
and Adipose Tissue

• Acetyl-CoA is the immediate substrate, and free

Palmitate (16 C) is the end product

7
Production of Cytosolic Acetyl-CoA

• The first step in de novo fatty acid synthesis is

the transfer of acetyl units from mitochondrial
acetyl CoA to the cytosol

• Mitochondrial acetyl CoA is produced by the

oxidation of pyruvate, and by the catabolism of
fatty acids, ketone bodies, and certain amino
acids
8
• The coenzyme A portion of acetyl CoA,
however, cannot cross the mitochondrial
membrane; only the acetyl portion is
transported to the cytosol

• It does so in the form of citrate produced by

the condensation of oxaloacetate (OAA) and
acetyl CoA (enzyme: citrate synthase)

9
10
• Citrate is transported from mitochondria by a
tricarboxylic acid transporter.

• In the cytoplasm, citrate is cleaved to oxaloacetate

and acetyl CoA in the cytoplasm in the presence of
ATP (enzyme: ATP citrate lyase).

 Thus, by the coordinated action of two enzymes–

citrate-synthase and -lyase, the acetyl CoA is
effectively transported from mitochondrial matrix to
the cytosol.
11
12
• While Acetyl CoA is used for fatty acid synthesis,
oxaloacetate is shuttled back into the mitochondrion
either as malate or as pyruvate.

• It is converted to malate via malate dehydrogenase

that uses NADH as the proton donor.

• Malate is then converted to pyruvate by the NADP-

dependent malic enzyme.

13
• The reduced NADPH so formed becomes

available for lipogenesis.

• Pyruvate is then translocated across the

mitochondrial membrane to be cycled again

14
• Cytosolic citrate may be viewed as a

high-energy signal

• Because a large amount of ATP is needed

for fatty acid synthesis, the increase in both

ATP and citrate enhances this pathway

15
 Production of Malonyl-CoA is the initial &
controlling Step in Fatty acid Synthesis

• Bicarbonate as a source of CO2 is required in the initial

reaction for the carboxylation of acetyl-CoA to malonyl-
CoA in the presence of ATP and acetyl-CoA
carboxylase.

16
• Acetyl-CoA Carboxylase has a requirement for the B-
vitamin biotin and is a multienzyme protein containing

i. Biotin,

ii. Biotin carboxylase (E1),

iii. Biotin carboxyl carrier protein (BCP),

iv. Carboxyl transferase (E2), as well as a

v. Regulatory allosteric site.

• One subunit of the complex contains all the

components, and variable number of subunits form
polymers in the active enzyme
17
18
• Biotin is covalently linked to the BCP.

• The reaction proceeds in 2 steps.

1. In step 1, catalysed by E1, biotin is carboxylated

as it accepts a COO− group from HCO3 − and ATP
is used.

2. In step 2, catalyzed by E2, the COO− is transferred

to acetyl-CoA forming malonyl-CoA

19
Short-term regulation of acetyl CoA

carboxylase:

• This carboxylation is both the rate-limiting and

the regulated step in fatty acid synthesis

• The inactive form of acetyl CoA carboxylase

is a protomer (dimer)
20
• The enzyme undergoes
allosteric activation by
citrate, which causes dimers
to polymerize

• The enzyme can be

allosterically inactivated by
long-chain fatty acyl CoA
(the end product of the
pathway), which causes its
depolymerization

21
22
• A second mechanism of short-
term regulation is by reversible
phosphorylation (Covalent
modification)

• In the presence of counter

regulatory hormones, such as
epinephrine and glucagon,
acetyl CoA carboxylase is
phosphorylated and, thereby,
inactivated
23
• In the presence of insulin, acetyl CoA

carboxylase is dephosphorylated and,

thereby, activated

24
 Long-term Regulation of Acetyl CoA carboxylase

• Prolonged consumption of a diet containing excess

calories (particularly, high-calorie, high-carbohydrate

diets) causes an increase in acetyl CoA carboxylase

synthesis, thus increasing fatty acid synthesis

• Conversely, a low-calorie diet or fasting causes a

reduction in fatty acid synthesis by decreasing the

synthesis of acetyl CoA carboxylase

25
 The Fatty Acid Synthase Complex Is a Polypeptide
Containing Seven Enzyme Activities

• The remaining series of reactions of fatty acid

synthesis in eukaryotes is catalyzed by the
multifunctional, dimeric enzyme, fatty acid
synthase complex

• The complex is a dimer of two identical polypeptide

monomers, 1 and 2, each consisting of seven enzyme
activity domains and an acyl carrier protein (ACP)

26
• These domains are arranged in such a way that
they catalyze the successive steps in the fatty acid
synthesis cycle.

 The biosynthetic intermediates do not diffuse away

from the FAS complex but remain attached to the
terminal thiol (SH) group of an acyl carrier protein
(ACP), which passes them from one enzyme’s
active site to the next

27
28
29
30
The growing fatty acid chain is bound
covalently to two thiol groups (-SH) during its
synthesis.

• These groups, two in each FAS monomer, serve as

carriers of acyl groups

A. One of these, called the central thiol, is

contributed by Acyl Carrier Protein (ACP)

31
 Coenzyme A
(CoA- SH)

32
33
• ACP contains the vitamin pantothenic acid in the
form of 4'-phosphopantetheine

• The 4'-phosphopantetheine prosthetic group of ACP

is believed to serve as a flexible arm, tethering the
growing fatty acyl chain to the surface of the fatty
acid synthase complex and carrying the reaction
intermediates from one enzyme active site to the
next

• It is also a component of Coenzyme A

34
B. The second important thiol group, called the
peripheral thiol, is contributed by a cysteine
residue (Cys) of the enzyme 3-ketoacyl
synthase.

• The two types of thiol groups lie in close proximity

in the dimer, suggesting that they are present on
separate (subunits) that interact in a head-to-tail
manner

35
• The use of one multienzyme functional unit has the
advantages of:

I. Achieving the effect of compartmentalization

of the process within the cell without the
erection of permeability barriers

II. Synthesis of all enzymes in the complex is

coordinated since it is encoded by a single
gene

36
• Each subunit (or monomer) contains all the
seven enzyme activities, but the actual
functional unit consists of one-half of a
monomer interacting with the complementary
half of the other monomer.

Thus, two fatty acid chains can be

synthesized simultaneously

37
38
• The first two reactions (called priming

reactions) ensure that both the thiol groups

are ‘loaded’ with correct acyl groups, and the

remaining reactions called elongation steps

are involved in actual building of the fatty acid

chain
39
 Priming Reactions

• The two priming reactions are catalyzed by enzymes

acetyl transacylase and malonyl transacylase.

1. Acetyl CoA- ACP transacylase

– This enzyme catalyzes transfer of an acetyl group

from acetyl CoA to the thiol group furnished by ACP.

– Next, this two-carbon fragment is transferred to a

temporary holding site, the thiol group of a cysteine
residue on the enzyme (3-ketoacyl synthase)

40
41
2. Malonyl CoA-ACP transacylase

– The now-vacant ACP accepts a three-carbon

malonate unit from malonyl CoA catalyzed by
Malonyl CoA-ACP transacylase to form acetyl
(acyl)-malonyl ACP

42
• At this state, both central and peripheral-thiol
groups are charged by malonyl and an acetyl group
respectively.

• Since the two acyl groups are close to each other

on the enzyme complex, the stage is now set for
their condensation and further elongation steps

Elongation Steps

• Condensation, reduction, dehydration and

reduction are the four elongation steps
43
1. Condensation by 3-Ketoacyl ACP synthase
• The acetyl group attacks the methylene group of
the malonyl residue, catalyzed by 3-ketoacyl
synthase, and liberates CO2, forming 3-ketoacyl
ACP (acetoacetyl enzyme), thus freeing the
cysteine —SH group.

• The loss of free energy from the decarboxylation

drives the reaction forward

44
3-ketoacyl ACP
(acetoacetyl ACP) 45
2. Reduction by 3-Ketoacyl ACP reductase:

• The keto group is reduced by this enzyme to form an D-3-

hydroxyacyl ACP.

• The reducing power for this reaction is provided by NADPH.

• D configuration is used in synthesis; L configuration is used

in oxidation

46
Ester

47
3. Dehydration by D-3-Hydroxyacyl-ACP dehydratase

• The elements of water are now removed from C-2 and

C-3 of D-3-hydroxyacyl ACP to yield a double bond in
the product, 2,3-Unsaturated acyl enzyme (trans-Δ2-
butenoyl ACP)

48
4. Reduction by enoyl-ACP reducatse

• The double bond is reduced.

Four-carbon, saturated fatty acyl-ACP (butyryl-ACP)

49
• This final reaction of elongation produces the four
carbon butyryl-ACP; thus the acyl group has grown
longer by two carbon atoms

• All the above enzymatic steps are carried out in

different enzyme domains of fatty acid synthase
with the flexible arm of the phosphopantetheine
group (of ACP) moving the acyl group from one
active site to the next.

50
• The result of these steps is production of a four-carbon
compound (butyryl) whose three terminal carbons are fully
saturated, and which remains attached to the ACP.

• These steps are repeated, beginning with the transfer of the

butyryl chain from the ACP to the Cys residue [2*], the
attachment of a molecule of malonate to the ACP [3*], and
the condensation of the two molecules liberating CO2 [4*].

• The carbonyl group at the β-carbon (carbon 3—the third

carbon from the sulfur) is then reduced [5*], dehydrated [6*],
and reduced [7*], generating hexanoyl-ACP

51
3-ketoacyl ACP

52
• The sequence of reactions is repeated five
more times until a saturated 16-carbon acyl
radical (palmitate) has been assembled

• With each passage through the cycle, the

fatty acyl chain is extended by two carbons

53
54
All the carbons in palmitic acid have passed
through malonyl CoA except the two donated
by the original acetyl CoA, which are found at
the methyl-group end of the fatty acid

This underscores the rate-limiting nature of

the acetyl CoA carboxylase reaction

The acetyl-CoA used as a primer forms

carbon atoms 15 and 16 of palmitate
55
• Palmitate is liberated from the enzyme
complex by the activity of a seventh enzyme
in the complex, thioesterase (deacylase)

The free palmitate must be activated to acyl-

CoA before it can proceed via any other
metabolic pathway

56
• Its usual fate is:

I. Esterification into Acylglycerols

II. Chain Elongation

III. Chain Desaturation

IV. Esterification to Cholesteryl Ester

57
• In mammary glands, there is a separate

thioesterase specific for acyl residues of C8,

C10, or C12, which are subsequently found in

milk lipids

58
• The biosynthesis of fatty acids such as
palmitate thus requires acetyl-CoA and the
input of chemical energy in two forms:

I. ATP is required to attach CO2 to acetyl-

CoA to make malonyl-CoA

II. The reducing power of NADPH is required

to reduce the double bond

59
• There is an additional cost to fatty acids
synthesis because acetyl-CoA is generated in
the mitochondria and must be transported to
the cytosol

• This extra step consumes two ATPs per

molecule of acetyl-CoA transported, increasing
the energetic cost of fatty acid synthesis to
three ATPs per two-carbon unit

60
61
Expenditure of ATP per 2-carbon unit
addition in fatty acid synthesis

1. Citrate Lyase

2. Pyruvate Carboxylase

3. Acetyl CoA Carboxylase

62
• In most higher eukaryotes, the fatty acid
synthase complex is found exclusively in the
cytosol as are the biosynthetic enzymes for
nucleotides, amino acids, and glucose

This location segregates synthetic processes

from degradative reactions, many of which
take place in the mitochondrial matrix

63
There is a corresponding segregation
of the electron-carrying cofactors used
in anabolism (generally a reductive
process) and those used in catabolism

(generally oxidative)

64
• Usually, NADPH is the electron carrier for
anabolic reactions (in the cytosol), and NAD+
serves in catabolic reactions (in the
mitochondrial matrix)

• In hepatocytes, the [NADPH]/[NADP+] ratio is

very high in the cytosol, furnishing a strongly
reducing environment for the reductive
synthesis of fatty acids and other biomolecules

65
The Main Source of NADPH for Lipogenesis
is the Pentose Phosphate Pathway

• NADPH is involved as donor of reducing

equivalents in both of the reduction reactions

• The reactions of the pentose phosphate

pathway are the chief source of the hydrogen
required for the reductive synthesis of fatty
acids

66
• Significantly, tissues specializing in active
lipogenesis—i.e. liver, adipose tissue, and the
lactating mammary gland—also possess an active
pentose phosphate pathway.

• Moreover, both metabolic pathways are found in

the cytosol of the cell

• So, there are no membranes or permeability

barriers against the transfer of NADPH

67
• Other source of NADPH include:

– The reaction that converts malate to

pyruvate catalyzed by the "malic

enzyme" (decarboxylating NADP malate

dehydrogenase)

68
 Elongation of Fatty Acid Chains Occurs in the Smooth
Endoplasmic Reticulum (SER)

• The "microsomal system” elongates saturated and

unsaturated fatty Acyl-CoAs (from C10 upward) by two
carbons, using malonyl-CoA as the acetyl donor and
NADPH as the reductant, and is catalyzed by the
microsomal fatty acid elongase system of enzymes

• The more active elongation system of the SER extends the

16-carbon chain of palmitoyl-CoA by two carbons, forming
stearoyl-CoA (18 C).

69
• Although different enzyme systems are involved, and

Co-enzyme A rather than ACP is the acyl carrier in the

reaction, the mechanism of elongation in the ER is

otherwise identical to that in palmitate synthesis:

donation of two carbons by malonyl-CoA, followed by

reduction, dehydration, and reduction to the saturated

18-carbon product, stearoyl-CoA.

70
• Elongation of stearyl-CoA in brain increases

rapidly during myelination in order to provide

C22 and C24 fatty acids for sphingolipids

71
 Monounsaturated Fatty Acids Are
Synthesized by a 9 Desaturase System

• Enzymes (desaturases) also present in the SER

are responsible for desaturating long-chain fatty
acids (that is, adding cis double bonds)

• The first double bond introduced into a saturated

fatty acid is nearly always in the 9 position

72
• An enzyme system—9 desaturase (a
mixed-function oxidase) in the endoplasmic
reticulum catalyzes the conversion of
palmitoyl-CoA or stearoyl-CoA to
palmitoleoyl-CoA or oleoyl-CoA, respectively

• Oxygen and either NADH or NADPH are

necessary for the reaction

73
• Humans have carbon 9, 6, 5 and 4 desaturases,
but lack the ability to introduce double bonds from
carbon 10 to the ω end of the chain.

• This is the basis for the nutritional essentiality of

the polyunsaturated linoleic and linolenic acids

• We obtain ω-6 and ω-3 polyunsaturated fatty acids

mainly from dietary plant oils that contain the ω-6
fatty acid Linoleic acid (18:2, Δ9,12) and the ω-3
fatty acid α-Linolenic acid (18:3, Δ 9,12,15)
74
• In the body, linoleic acid can be converted by

elongation and desaturation reactions to ω-6

fatty acid Arachidonic acid (20:4, Δ5,8,11,14),

which is used for the synthesis of the major

class of human prostaglandins and other

eicosanoids
75
76
Synthesis of Polyunsaturated Fatty Acids
Involves Desaturase & Elongase Enzyme
Systems

• Additional double bonds introduced into

existing monounsaturated fatty acids are
always separated from each other by a
methylene group (methylene interrupted)

77
• If the fatty acid has
two or more double
bonds they are
always spaced at
three carbon
intervals

78
 Biosynthesis of Triacylglycerols (TAGs)

• Most of the fatty acids synthesized or ingested by an

organism have one of two fates:

i. Incorporation into triacylglycerols for the storage

of metabolic energy

ii. Incorporation into the phospholipid components

of membranes

• Both pathways begin at the same point:

the formation of fatty acyl esters of glycerol

79
• The partitioning between these alternative fates
depends on the organism's current needs:

a. During rapid growth, synthesis of new

membranes requires the production of
membrane phospholipids

b. When an organism has a plentiful food supply

but is not actively growing, it shunts most of its
fatty acids into storage fats

80
• Mono-, di-, and triacylglycerols consist of one, two,

or three molecules of fatty acid esterified to a

molecule of glycerol.

• Fatty acids are esterified through their carboxyl

groups, resulting in a loss of negative charge and

formation of “neutral fat”.

81
82
Ester

83
1. Structure of Triacylglycerol (TAG)

• The three fatty acids esterified to a glycerol molecule are

usually not of the same type.

• The fatty acid on carbon 1

is typically saturated, that

on carbon 2 is typically

unsaturated, and that on

carbon 3 can be either

84
• Both glycerol and fatty acids must be activated by ATP
before they can be incorporated into acylglycerols.

• Glycerol kinase catalyzes the activation of glycerol to

glycerol 3-phosphate (in liver).

• If the activity of this enzyme is absent or low, as in

muscle or adipose tissue, most of the glycerol-3-
phosphate is formed from dihydroxyacetone phosphate
by glycerol-3-phosphate dehydrogenase

85
2. Synthesis of Glycerol Phosphate

• Glycerol phosphate is the acceptor of fatty acids during

TAG synthesis

• There are two pathways for glycerol phosphate production

1. The liver, but NOT adipose tissue, uses glycerol

kinase to convert free glycerol to glycerol phosphate

2. In both liver (the primary site of TAG synthesis) and

adipose tissue, glycerol phosphate can be produced
from glucose

86
• Glycerol phosphate can be produced from glucose,
using first the reactions of the glycolytic pathway to
produce dihydroxyacetone phosphate (DHAP).

• Next, DHAP is reduced by glycerol phosphate

dehydrogenase to glycerol phosphate by cytosolic
NAD-linked glycerol 3-phosphate dehydrogenase

87
Pathways for production of glycerol phosphate in liver
and adipose tissue

88
3. Conversion of a free fatty acid to its activated form

• The other precursors of triacylglycerols are fatty acyl-

CoAs

• A fatty acid must be converted to its activated form

before it can participate in metabolic processes
such as TAG synthesis.

• They are activated by being joined in thioester

linkage (R-CO-SCoA) to the -SH group of
coenzyme A.
89
This reaction is catalyzed by a family of fatty
acyl CoA synthetases (thiokinases)

90
Ester

91
4. Synthesis of Phosphatidic Acid

• Glycerol 3-P is then acylated by transfer of two long

chain fatty acids from fatty acyl CoA to the hydroxyl
groups at C-1 and C-2 producing phosphatidic acid.

• The first fatty acid, usually a saturated fatty acid, is

linked to C-1 forming monoacyl-glycerol-P, also
called lysophosphatidic acid; the prefix lyso
indicates that one of the hydroxyl groups is not
acylated.
92
• Then a second fatty acid, usually an

unsaturated fatty acid, establishes an ester

bond to C-2 to form phosphatidic acid

93
94
• Two different enzymes catalyze these two
acylation steps—
glycerol phosphate acyl transferase (E) and
monoacyl glycerol acyl transferase (E’)

95
5. Synthesis of triacylglycerol from phosphatidic
acid

• Phosphatidic acid is first converted to diacyglycerol

(DAG) by a specific phospatidic acid phosphatase,
which removes a phosphate group.

• Transfer of an acyl group from fatty acyl CoA to

DAG by the enzyme 1,2-Diacylglycerol acyl
transferase finally generates triacylglycerol (TAG).

96
97
• Note that the ester bonds of triacylglycerol
are low energy in nature.

• Fatty acyl CoA, the acyl group donor, on the

other hand, has a high energy thioester.

• Therefore, the acyl transfer reactions

proceed with a decrease in the number of
high energy bonds, and are therefore
exergonic
98
Different fates of TAG in the liver and
adipose tissue

1. Fate In Liver: Little TAG is stored in the liver

• Instead, most is exported, packaged with

cholesteryl esters, cholesterol, phospholipid,
and protein to form lipoprotein particles called
very low density lipoproteins (VLDL)

99
100
• Nascent VLDL are secreted into the blood where
they mature and function to deliver the
endogenously-derived lipids to the peripheral
tissues

2. Fate in Adipose Tissue: In adipose tissue, TAG

is stored in the cytosol of the cells in a nearly
anhydrous form

• It serves as "depot fat," ready for mobilization

when the body requires it for fuel
101
• Neutral lipids are stored in adipocytes (and in
steroid synthesizing cells of the adrenal cortex,
ovary and testes) in the form of lipid droplets, with
a core of sterol esters and triacylglycerols
surrounded by a monolayer of phospholipids

 The surface of these droplets is coated with

perilipins, a family of proteins that restrict access
to lipid droplets, preventing untimely lipid
mobilization
102
103
RELEASE OF FATTY ACIDS FROM ADIPOSE
TRIACYLGLYCEROLS

• During fasting, the decrease of insulin and the

increase of glucagon (or epinephrine) cause cAMP
levels to rise in adipose cells, stimulating lipolysis

• The increased level of cyclic AMP then stimulates

protein kinase A, which phosphorylates two key
proteins: Perilipin, a fat-droplet-associated protein,
and Hormone-sensitive Lipase.

104
• The phosphorylation of Perilipin has crucial effects:

I. First, it restructures the fat droplet so that the

triacylglycerols are more accessible to the
mobilization.

II. Second, the phosphorylation of perilipin triggers

the release of a coactivator for the adipose
triglyceride lipase (ATGL). Once bound to the co
activator, ATGL initiates the mobilization of
triacylglycerols by releasing a fatty acid from
triacylglycerol, forming diacylglycerol.
105
 – Diacylglycerol is converted into a free fatty acid
and monoacylglycerol by the hormone-sensitive
lipase

III. The phosphorylated perilipin also causes

hormone sensitive lipase in the cytosol to move
to the lipid droplet surface where it can begin
hydrolyzing triacylglycerols to free fatty acids and
glycerol

106
 PKA also phosphorylates hormone-sensitive lipase,
doubling or tripling its activity, but more than 50-fold
increase in fat mobilization triggered by epinephrine
is primarily due to the perilipin phosphorylation.

• Cells with defective perilipin genes have almost no

response to increases in cAMP concentration and
their hormone-sensitive lipase does not associate
with lipid droplets

107
108
109
• Subsequently, other lipases complete the process

of lipolysis, and fatty acids and glycerol are

released into the blood

 Thus, epinephrine and glucagon induce lipolysis

and insulin inhibits it

110
 Fate of Free Fatty Acids & Glycerol

A. Fate of Free Fatty Acids

• The released fatty acids are not soluble in blood

plasma, and so the blood protein albumin binds the

fatty acids and serves as a carrier.

• By these means, free fatty acids are made accessible

as a fuel in other tissues.

111
• Some of the fatty acids released by lipolysis of
triacylglycerol in adipose tissue pass in to the
bloodstream, and the remainder are used for
resynthesis of triacylglycerol

• Some of the fatty acids released in to the blood are

used for energy (in muscle, for example) and some
are taken up by the liver and used in triacylglycerol
synthesis

112
• This triacylglycerol cycle is an additional factor in
the balance between biosynthesis and degradation
of triacylglycerols in that approximately 75% of all
fatty acids released by lipolysis are re-esterified to
form triacylglycerols rather than used for fuel

• This ratio persists even under starvation conditions,

when energy metabolism is shunted from the use of
carbohydrate to the oxidation of fatty acids

113
• The triacylglycerol
formed in the liver is
transported in the
blood back to adipose
tissue, where the fatty
acid is released by
extracellular lipoprotein
lipase, taken up by
adipocytes, and re-
esterified in to
triacylglycerol
114
B. Fate of Glycerol

• Because adipose tissue lacks glycerol kinase and

cannot use the glycerol produced by LPL (& HSL),
the glycerol travels through the blood to the liver
where it is phosphorylated.

• It is then oxidized to dihydroxyacetone phosphate,

which is isomerized to glyceraldehyde 3-
phosphate.

115
• This molecule is an intermediate in both the glycolytic
and the gluconeogenic pathways

• Hence, glycerol can be converted into pyruvate or

glucose in the liver, which contains the appropriate
enzymes.

• The reverse process can take place by the reduction of

dihydroxyacetone phosphate to glycerol 3-phosphate.

• Thus, glycerol and glycolytic intermediates are readily

interconvertible.

116
117
118
Hormonal Regulation of Adipose Tissue
Metabolism

• The metabolic processes operative within

adipocytes are regulated by a number of hormones
which control activities of both the key enzymes of
adipose tissue metabolism, namely, the Hormone-
sensitive Lipase and the Glycerol Phosphate
Acyl Transferase

119
• The enzyme activity is regulated by the cAMP-
dependent cascade; activation occurs when the
enzyme is phosphorylated by the cAMP-dependent
protein kinase-A (PKA).

• Thus, the hormones which increase intracellular

level of cAMP and activate the hormone sensitive
lipase are lipolytic

120
121
• The most important lipolytic agents are

catecholamines which act through β-adrenergic

receptors, cAMP and protein kinase A (PKA).

• The catecholamines ensure that the TAGs are

hydrolyzed during cold exposure, stress and

physical exercise through stimulation of the lipase

activity
122
• Glucocorticoids, growth hormone and the thyroid
hormones enhance lipolysis by inducing synthesis
of lipolytic proteins.

• Thus, glucocorticoids induce the synthesis of the

hormone-sensitive lipase, and thereby accelerate
the lipolytic response to catecholamines

 In short, Lipolytic hormones facilitate lipolysis either

by direct stimulation of cAMP formation or by
inducing synthesis of lipolytic protein (HSL).
123
Role of Insulin

• In contrast to the lipolytic hormones-action of

insulin is antilipolytic, i.e. it antagonizes these
hormones by causing inhibition of the hormone-
sensitive lipase.

• The mechanism of action is poorly understood.

• Either insulin decreases the level of cAMP in the

cell (by activating a phosphodiesterase) or induces
dephosphorylation of the HSL (by phosphatase).
124
125
• Adipose tissue is highly sensitive to insulin: a
moderate rise in insulin level (during feeding)
is able to halt lipolysis.

Insulin enhances TAG synthesis

• In addition to inhibiting lipolysis, insulin can

also enhance TAG synthesis in adipocytes
by several mechanisms:

126
1. By increasing production of glycerol 3-
phosphate substrate:

i. Insulin increases the rate of glycolysis, which

enhances production of dihydroxyacetone
phosphate which is then reduced to glycerol 3-
phosphate

ii. Insulin enhances the glucose uptake by

increasing the number of functional glucose
carriers in plasma membrane.
127
2. By increasing synthesis of fatty acid substrate:

i. It increases production of acetyl CoA from glucose

(by augmenting glycolytic sequence), and acetyl CoA
is the substrate for lipogenesis.

ii. Further, it stimulates activity of acetyl CoA

carboxylase, the key enzyme of lipogenesis.

iii. Insulin favors generation of NADPH, the coenzyme

required in lipogenesis, by promoting the pentose
phosphate pathway (upregulates expression of G6
PD gene).
128
3. By increasing lipoprotein lipase activity:

• Stimulation of activity of this enzyme results in

increased production of fatty acids from the TAG
fraction of lipoprotein.

• This further increases the supply of fatty acid substrate.

4. By increasing esterification of fatty acid and

glycerol substrates:

• Insulin induces the enzyme glycerol phosphate acyl

transferase which adds the first fatty acid to glycerol
phosphate in the bio-synthetic pathway of TAG
129
• Thus, insulin has a dual role—inhibition of lipolysis
and stimulation of triacylglycerol production.

• This helps to maintain a balance between these two

pathways, under normal circumstances.

• In diabetes mellitus, loss of this balance occurs.

• Consequently, lipolysis is accelerated, but

triacylglycerol synthesis slows.

• The net result is that the rate of lipolysis now

exceeds that of triacylglycerol synthesis.
130
• Quantity of fatty acids produced is far in excess than

that can be reutilized for re-esterification.

• The surplus flows out into the circulation to elevate the

plasma levels of free fatty acids.

• This explains a marked elevation of plasma free fatty

acid levels in diabetes mellitus and loss in weight of the

patient

131
Nutritional regulation of Adipose Tissue
Metabolism

• The metabolic events in adipocytes show dramatic

changes in different nutritional states.

• TAG synthesis depends on the supply of glycerol

phosphate, which is synthesized from glucose via
dihyroxyacetone phosphate

• The insulin-dependent uptake of glucose into the

cell is an important step in this pathway.
132
• Thus, in the fed state, increased availability of
glucose and elevated insulin level in serum
enhance TAG synthesis.

• Conversely, during starvation synthesis of TAG is

impeded

• Insulin (& glucagon to a lesser extent) ensures that

triacylglycerols are synthesized after a meal, and
degraded during fasting.

133
 Oxidation of Fatty Acids

• The complete oxidation of fatty acids to CO2 and H2O

takes place in three stages:

1. The oxidation of long-chain fatty acids to two-

carbon fragments, in the form of acetyl-CoA (β
oxidation)

2. The oxidation of acetyl-CoA to CO2 in the citric

acid cycle

3. The transfer of electrons from reduced electron

carriers to the mitochondrial respiratory chain
134
135
• Here, we focus on the first of these stages

 We begin our discussion of β oxidation with the

simple case in which a fully saturated fatty acid with
an even number of carbon atoms is degraded to
acetyl-CoA

• We then Iook briefly at the extra transformations

necessary for the degradation of unsaturated fatty
acids and fatty acids with an odd number of carbons
136
• Finally, we discuss variations on the β-oxidation theme

– Two Iess common pathways of fatty acid

catabolism,

• ω oxidation

• α oxidation

• This discussion concludes with a description of an

alternative fate for the acetyl-CoA formed by β
oxidation in vertebrates:

– The production of ketone bodies in the liver

137
Fatty Acid Catabolism

• It consists of repeated cycles of a series of

reactions.

• With each cycle, a two-carbon unit (i.e. acetyl CoA

molecule) is removed from the carboxyl terminal of
the fatty acid.

138
• Thus complete oxidation of the 16-carbon fatty acid
(e.g. palmitic acid) requires seven such cycles and
generates eight molecules of acetyl CoA.

• The oxidation of long-chain fatty acids to acetyl-

CoA is a central energy-yielding pathway in many
organisms and tissues

• In mammalian heart and liver, for example, it

provides as much as 80% of the energy needs
under all physiological circumstances
139
• The electrons removed from fatty acids
during oxidation pass through the respiratory
chain, driving ATP synthesis

• The acetyl-CoA produced from the fatty acids

may be completely oxidized to CO2 in the
citric acid cycle, resulting in further energy
conservation. (Citric acid cycle also occur in
mitochondria)
140
• In some species and in some tissues, the

acetyl-CoA has alternative fates

• In liver, acetyl-CoA may be converted to

ketone bodies, which are water-soluble fuels

exported to the brain and other tissues

when glucose is not available

141
• The Oxidative pathway of fatty acids occurs

in three stages:

A. Activation of fatty acid in the cytosol

B. Transport of activated fatty acid into

mitochondria

C. Standard β-oxidation process in

mitochondrial matrix
142
A. Activation of Fatty Acid

• As the priming step for catabolism, the fatty acids

are activated through formation of a thioester
linkage between the carboxyl group of the fatty acid
molecule and thiol group of coenzyme A.

• The product is an acyl coenzyme A, and therefore

the name of the enzyme that catalyzes this reaction
is acyl CoA synthetase, also called thiokinase.

143
• This reaction requires a great deal of energy
and in the process ATP is converted to AMP

• This was the first reaction in biochemistry found to

yield pyrophosphate by cleavage of ATP

i. The carboxylate ion is adenylylated by ATP, to

form a fatty acyl-adenylate (intermediate) and PPi

ii. The thiol group of coenzyme A attacks the acyl-

adenylate, displacing AMP and forming the fatty
acyl-CoA
144
AMP= 5’adenylic acid; substituent= Adenylyl-

145
146
• These partial reactions are freely reversible. In fact,
the equilibrium constant for the sum of these
reactions is close to 1

• One high energy bond is cleaved (between PPi and

AMP) and one high energy bond is formed (the
thioester acyl CoA).

• How is the overall reaction driven forward? The

answer is that pyrophosphate is rapidly hydrolyzed
by a pyrophosphatase
147
• This reaction is quite favorable because the
equivalent of two high energy bonds are
hydrolyzed, whereas only one high energy bond is
formed.

• We see here another example of a recurring theme

in biochemistry

Many biosynthetic reactions are made irreversible

by the hydrolysis of inorganic pyrophosphate.

148
• There are three different thiokinases for
different fatty acids:

i. One for Short chain fatty acids,

ii. One for Medium chain,

iii. One for Long chain and Very long chain

fatty acids

149
B. Transport of activated fatty acid into
mitochondria

• Fatty acyl-CoA esters formed at the cytosolic side of

the outer mitochondrial membrane can be:

I. Transported into the mitochondrion and

oxidized to produce ATP

II. Used in the cytosol to synthesize membrane

lipids

III. Used in the formation of TAGs

150
• The enzymes of fatty acid oxidation in animal cells
are located in the mitochondrial matrix

• The fatty acids with chain lengths of 12 or fewer

carbons enter mitochondria without the help of
membrane transporters

• Those with 14 or more carbons, which constitute

the majority of the FFA obtained in the diet or
released from adipose tissue, cannot pass directly
through the mitochondrial membranes
151
• They must first undergo the three enzymatic reactions
of the CARNITINE SHUTTLE

• These fatty acids must be conjugated to carnitine, a

zwitterionic alcohol.

• The acyl group is transiently transferred from the sulfur

atom of coenzyme A to the hydroxyl group of carnitine
to form acyl carnitine.

• This is catalyzed by Carnitine AcylTransferase I (CAT

I aka CPT I), in the outer mitochondrial membrane

152
Transestrification

153
In organic chemistry, trans-esterification is the
process of exchanging the organic group R’’ of
an ester with the organic group R’ of an alcohol.

154
155
• Passage of the carnitine ester into the
intermembrane space occurs through large
pores (formed by the protein porin) in the
outer membrane

• The fatty acyl-carnitine ester then enters the

matrix by facilitated diffusion through the
acylcarnitine/carnitine translocase of the
inner mitochondrial membrane
156
157
158
159
• In the final step of the carnitine shuttle, the

fatty acyl group is enzymatically transferred

from carnitine to intramitochondrial coenzyme A

by Carnitine Acyltransferase II (CAT II)

• This is simply the reverse of the reaction that

took place in the cytoplasm.

160
• This isozyme, Iocated on the inner face of the inner

mitochondrial membrane, regenerates fatty acyl-

CoA and releases it, along with free carnitine, into

the matrix

• Finally, the translocase returns carnitine to the

cytoplasmic side in exchange for an incoming acyl

carnitine.
161
• The carnitine-mediated entry process is the
rate Iimiting step for oxidation of fatty acids
in mitochondria

• Once inside the mitochondrion, the fatty acyl-

CoA is acted upon by a set of enzymes in the
matrix for fatty acid oxidation

162
Inhibitor of the Carnitine Shuttle

• Malonyl CoA inhibits CAT-I, thus preventing the

entry of long-chain acyl groups into the
mitochondrial matrix

• Therefore, when fatty acid synthesis is occurring in

the cytosol (as indicated by the presence of malonyl
CoA), the newly made palmitate cannot be
transferred into the mitochondria and degraded
163
Sources of Carnitine

• Carnitine can be obtained from the diet,

where it is found primarily in meat products

• Carnitine can also be synthesized from the

amino acids lysine and methionine by an
enzymatic pathway found in the liver and
kidney but not in skeletal or heart muscle

164
Carnitine Deficiencies:

• Such deficiencies result in:

a. Decreased ability of tissues to use

LCFA as a metabolic fuel.

b. Accumulation of toxic amounts of

free fatty acids and branched-chain
acyl groups in cells
165
 Secondary Carnitine Deficiency occurs for many reasons,
including

i. In patients with liver disease causing decreased

synthesis of carnitine

ii. In individuals suffering from malnutrition or those on

strictly vegetarian diets

iii. In those with an increased requirement for carnitine as a

result of, for example, to pregnancy, severe infections,
burns, or trauma

iv. In those undergoing hemodialysis, which removes

carnitine from the blood
166
Congenital deficiencies in

i. One of the components of the carnitine

acyltransferase system

ii. Tubular reabsorption of carnitine

iii. Carnitine uptake by cells

167
Genetic CAT I deficiency

• It affects the liver, where an inability to use

LCFA for fuel greatly impairs that tissue's
ability to synthesize glucose during a fast

• This can lead to severe hypoglycemia, coma,

and death

168
CAT II deficiency

• It occurs primarily in cardiac and skeletal

muscle, where symptoms of carnitine
deficiency range from cardiomyopathy, to
muscle weakness

• This is an example of how the impaired flow

of a metabolite from one cell compartment to
another results in pathology
169
Treatment includes:

– Avoidance of prolonged fasts

– Adopting a diet high in carbohydrate and

low in LCFA

– Diet supplemented with MCFA

– Diet supplemented with carnitine, in cases

of carnitine deficiency

170
Entry of short- and medium-chain fatty acids
into the mitochondria

• Fatty acids shorter than twelve carbons can cross

the inner mitochondrial membrane without the aid of
carnitine or the CAT system

• MCFAs are plentiful in human milk

• Because their oxidation is not dependent on CAT I,

it is not subject to inhibition by malonyl CoA

171
 Reactions of β-Oxidation

• The standard β-oxidation process comprises a series of

identical cycles.

• Each cycle shortens the fatty acid by two carbons and

produces an acetyl residue in the form of acetyl CoA.

• Repeated cycles of β-oxidation generate several acetyl CoA

molecules which enter TCA cycle and are further degraded
to carbon dioxide and water.

• The reaction sequence is called β-oxidation because it is the

β-carbon (C-3) that is oxidized

172
 The β-Oxidation of Saturated Fatty Acids Has Four
Basic Steps

• Four enzyme-catalyzed reactions make up the first

stage of fatty acid oxidation

• These steps include:

1. An oxidation that produces FADH2

2. A hydration step

3. A second oxidation that produces NADH+H+

4. A thiolytic cleavage that releases a molecule of

Acetyl CoA
173
174
1. First Oxidation
(dehydrogenation)

• A pair of hydrogen atoms

is removed from the α-
and β-carbons of the fatty
acyl CoA by action of fatty
acyl CoA dehydrogenase
resulting in the formation
of trans-Δ2-enoyl CoA
175
• As in the dehydrogenation of succinate in the citric
acid cycle, FAD rather than NAD+ is the electron
acceptor because the ΔG for this reaction is
insufficient to drive the reduction of NAD+.

• The reduced form of the dehydrogenase immediately

donates its electrons to an electron carrier of the
mitochondrial respiratory chain, the electron-
transferring flavoprotein (ETF)

176
• This first step is catalyzed by three isozymes of acyl-
CoA dehydrogenase, each specific for a range of
fatty-acyl chain lengths:

– Very long-chain acyl-CoA dehydrogenase

(VLCAD), acting on fatty acids of 12 to 18 carbons

– Medium-chain (MCAD), acting on fatty acids of 6

to 12 carbons

– Short-chain (SCAD), acting on fatty acids with less

than 6 carbons
177
Medium-chain fatty acyl CoA dehydrogenase
(MCAD) deficiency
• MCAD deficiency, an autosomal, recessive disorder, is one of
the most common inborn error of metabolism, and the most
common inborn error of fatty acid oxidation, being found
in 1 in 12,000 births in the west, and 1 in 40,000 worldwide

• It causes a decrease in fatty acid oxidation and severe

hypoglycemia (because the tissues cannot obtain full
energetic benefit from fatty acids and, therefore, must now
rely on glucose)
178
• Treatment includes a carbohydrate-rich diet

• Infants are particularly affected by MCAD deficiency,

because they rely for their nourishment on milk,
which primarily contains MCADs

• MCAD dehydrogenase deficiency has been identified

as the cause of some cases originally reported as
sudden infant death syndrome (SIDS)

179
2. Hydration

• Water is added to the double bond of the trans-2

enoyl- CoA to form the L stereoisomer of β-
hydroxyacyl-CoA.

• The reaction is catalyzed by enoyl CoA hydratase.

• In fact there are two hydratases: one preferring

short chain fatty acids and the second long chain.

• Both are highly specific and act only on the trans

isomer (i.e. trans-Δ2- enoyl CoA).
180
181
3. Second Oxidation
(Dehydrogenation)

• In the third step, L-β-

carbon looses a pair of
hydrogen atoms to form β-
ketoacyl-CoA, by the
action of β-hydroxyacyl-
CoA dehydrogenase

• NAD+ is the electron

acceptor
182
• Thus, by a sequential action of three enzymes, a
keto group is introduced at C-3 (β) position of the
fatty acyl CoA molecule

183
184
4. Thiolytic Cleavage

• The last step of the cycle is

catalyzed by acyl-CoA
acetyl transferase, aka β-
keto-thiolase, which
promotes reaction of β-
ketoacyl-CoA with a
molecule of free Coenzyme
A to split off the carboxyl-
terminal two-carbon
fragment of the original fatty
acid as acetyl-CoA 185
• The other product is the coenzyme A thioester of

the fatty acid, now shortened by two carbon atoms

• This reaction is called thiolysis, by analogy with

the process of hydrolysis, because the β-ketoacyl-

CoA is cleaved by reaction with the thiol group of

coenzyme A

186
• In each reaction cycle, an acyl CoA is shortened by
two carbon atoms, and one molecule each of
FADH2, NADH+H+, and acetyl CoA are formed.

• Following removal of one acetyl-CoA unit from

palmitoyl-CoA, the coenzyme A thioester of the
shortened fatty acid (now the l4-carbon myristate)
remains

187
• The myristoyl-CoA can now go through another set
of four β oxidation reactions, exactly analogous to
the first, to yield a second molecule of acetyl-CoA
and lauroyl-CoA, the coenzyme A thioester of the
l2-carbon laurate

• Altogether, seven passes through the β-oxidation

sequence are required to oxidize one molecule of
palmitoyl-CoA to eight molecules of acetyl-Co A

188
• These four steps are
repeated for saturated fatty
acids of even-numbered
carbon chains (n/2)-1 times
(where n is the number of
carbons), each cycle
producing an acetyl group
plus one NADH+H+ and one
FADH2

• The final thiolytic cleavage

produces two acetyl groups

189
Energy yield from Fatty Acid Oxidation

• The energy yield from the β-oxidation pathway is

high

• Each molecule of FADH2 formed during oxidation

of the fatty acid donates a pair of electrons to ETF
of the respiratory chain, and about 1.5 molecules of
ATP are generated during the ensuing transfer of
each electron pair to O2

190
191
192
193
• Each molecule of NADH+H+ formed delivers a pair

of electrons to the mitochondrial NADH

dehydrogenase, and results in formation of about

2.5 molecules of ATP

• Thus four molecules of ATP are formed for each

two-carbon unit removed in one pass through the

sequence, i.e. 28 molecules of ATP

194
• The acetyl-CoA produced from the oxidation of fatty
acids can be oxidized to CO2 and H2O by the citric
acid cycle, each acetyl CoA producing 10 ATP, i.e.
80 in all

• Thus, the oxidation of a molecule of palmitoyl CoA

to CO2 and H2O yields a total of:

20+80 =108 ATPs

195
• Because the activation of palmitate to
palmitoyl-CoA breaks both
phosphoanhydride bonds in ATP, the
energetic cost of activating a fatty acid is
equivalent to two ATP, therefore, the net gain
per molecule of palmitate is

108-2 = 106 ATP

196
 Saturation of Fatty Acids

• Fatty acid chains may contain no double bonds, that is be

saturated, or contain one or more double bonds that is, be
mono- or polyunsaturated

• When double bonds are present, they are nearly always in

the cis rather than in the trans configuration

197
• If the fatty acid has
two or more double
bonds they are
always spaced at
three carbon
intervals

198
Isomerism

• Two types of isomers can occur in an unsaturated

fatty acid

• They depend on the orientation of the radicals

around the axis of the double bonds

• If the hydrogen atoms are on the same side of the

bond, it is called as ‘cis’ form

• If the hydrogen atoms are on the opposite side, a

‘trans’ form is produced
199
200
Oxidation of Unsaturated Fatty Acids

• Approximately one half of the fatty acids in the

human diet are unsaturated, containing cis
double bonds, with oleate (C18:1, Δ9) and
linoleate (18:2, Δ9,12) being the most common

• In β-oxidation of saturated fatty acids, a trans

double bond is created between the 2nd and
3rd (α and β) carbons
201
Oxidation of Monounsaturated Fatty Acids

• The monounsaturated fatty acids, for example, oleic

acid (C18:1, Δ9), has 18 carbon atoms with a
cis double bond in position 9.

• After three β-oxidation cycles, the chain is

shortened by six carbons, and now the oxidative
mechanism encounters cis-Δ3-enoyl CoA

202
• This compound is not a natural substrate for β-
oxidation because there is a double bond between
C-3 and C-4 in cis configuration.

• For β-oxidation to proceed, we need a double bond

in trans- Δ2-configuration in enoyl CoA.

• This problem is solved by the action of an

isomerase (Δ3-cis-Δ2-trans-enoyl-CoA
isomerase) which converts the cis-Δ3 bond into
trans- Δ2 bond.
203
cis-Δ3 enoyl CoA

trans-Δ2 enoyl CoA

204
• As a result cis-Δ3-enoyl CoA is converted to

trans- Δ2-enoyl CoA.

• The latter being a normal substrate for β-

oxidation, the reaction cycle can now proceed

further

205
Oxidation of Polyunsaturated Fatty Acids
• Consider linoleate, a C18 polyunsaturated fatty
acid with cis-Δ9 and cis-Δ12 double bonds.

• The cis-Δ3 double bond (between carbons 3

and 4) formed after three rounds of β-oxidation
is converted into a trans-Δ2 double bond
(between carbons 2 and 3) by the
aforementioned isomerase.

206
 Cis Δ9,Δ12

Cis Δ3,Δ6

Cis Δ3 Enoyl CoA

trans Δ2,cisΔ6

207
 Cis Δ9,Δ12

Cis Δ3,Δ6

Cis Δ3 Enoyl CoA

trans Δ2,cisΔ6

Cis Δ4 trans Δ2,cisΔ4

208
• The acyl CoA produced by another round of
β-oxidation contains a cis-Δ4 (between
carbons 4 and 5) double bond.

• Dehydrogenation of this species by acyl CoA

dehydrogenase yields a 2,4-dienoyl
intermediate (trans Δ2, cis Δ4), which is not a
substrate for the next enzyme in the β-
oxidation pathway.
209
• This problem is solved by 2,4-dienoyl CoA

reductase, which uses reducing equivalants of

NADPH to convert the conjugated double bonds

(two double bonds separated by one single bond)

to a single Beta-gamma (carbons 3-4) bond.

210
• The Beta-gamma bond is flipped to the 2-3
position by Enoyl-CoA isomerase to form
trans-Δ2-enoyl-CoA, which is then
metabolized as earlier.

• Only two extra enzymes are needed for the

oxidation of any polyunsaturated fatty acid

211
 Cis Δ9,Δ12

Cis Δ3,Δ6

Cis Δ3 Enoyl CoA

trans Δ2,cisΔ6

Cis Δ4 trans Δ2,cisΔ4

212
Oxidation of fatty acids with an odd number of
carbons

• The β-oxidation of a saturated fatty acid with an odd

number of carbon atoms proceeds by the same
reaction steps as that of fatty acids with an even
number, until the final three carbons are reached

213
• This compound, propionyl CoA, is

metabolized by a three step pathway

• Propionyl CoA is also produced during the

metabolism of branched chain amino acids

214
1. Synthesis of D-methyl malonyl CoA

• First, propionyl CoA is carboxylated, forming D-

methylmalonyl CoA

• The enzyme propionyl CoA carboxylase has an absolute

requirement for the coenzyme biotin, as do other
carboxylases

215
216
2. Formation of L-
methylmalonyl CoA

• Next, the D-isomer

converted to the L-
form by the enzyme,
methylmalonyl CoA
racemase

217
3. Synthesis of
Succinyl CoA

• Finally, the carbons

of L-methyl malonyl
CoA are rearranged
by a mutase, forming
succinyl CoA, which
can enter the TCA
cycle 218
219
• The enzyme, methyl malonyl CoA mutase requires

a coenzyme form of vitamin B12 (deoxyadenosyl

cobalamin) for its action

• In patients with vitamin B12 deficiency, both

propionate and methyl malonate are excreted in

the urine

220
• Two types of inheritable methylmalonic acidemia
and aciduria have been described:

i. One in which the mutase is missing or deficient

(or has reduced affinity for the coenzyme)

ii. One in which the patient is unable to convert

vitamin B12 into its coenzyme form

• Either type results in metabolic acidosis, with

developmental retardation seen in some patients

221
• The propionyl CoA to succinyl CoA pathway
is a major anaplerotic route for the TCA cycle

• Succinyl CoA, an intermediate of the TCA

cycle, can form malate, which can be
converted to glucose in the liver through the
process of gluconeogenesis

222
223
• Thus, this small proportion of the odd-carbon-
number fatty acid chain can be converted to
glucose

• In contrast, the acetyl CoA formed from β-

oxidation of even-chain-number fatty acids in the
liver either enters the TCA cycle, where it is
principally oxidized to CO2, or is converted to
ketone bodies
224
Alternate Routes Of Fatty Acid Oxidation

• Mitochondrial β-oxidation can smoothly oxidize

saturated & unbranched fatty acids with an even
number of carbons and a chain length up to 18 or
20 carbons.

• Fatty acids that do not fit this description require

additional enzymatic reactions:

A. Unsaturated and odd chain fatty acids require

additional processing before they are β-oxidized
225
B. Very long chain fatty acids (VLCFA) are handled
initially by peroxisomal β-oxidation pathway.

C. Methylated fatty acids require α-oxidation initially,

before they can be β-oxidized.
D. Medium chain fatty acids can also be degraded by
(mostly β-oxidation) ω-oxidation.

• The function of these pathways is to convert as much

as possible of the unusual fatty acids to compounds
that can be used as fuels or biosynthetic precursors

226
Peroxisoms Also Carry Out β Oxidation

• The mitochondrial matrix is the major site of fatty

acid oxidation in animal cells, but in certain cells

other compartments also contain enzymes capable

of oxidizing fatty acids to acetyl-CoA, by a pathway

similar to, but not identical with, that in mitochondria

 In plant cells, the major site of β oxidation is not

mitochondria but peroxisomes

227
• In peroxisomes, the intermediates for β oxidation of
fatty acids are coenzyme A derivatives, and the
process consists of four steps, as in mitochondrial β
oxidation

i. Dehydrogenation (First Oxidation),

ii. Addition of water to the resulting double bond,

iii. Oxidation of the β -hydroxyacyl-CoA to a ketone

iv. Thiolytic cleavage by CoA-SH

228
1. One difference between the peroxisomal and

mitochondrial pathways lies in the chemistry of the

first step

• In peroxisomes, acyl CoA dehydrogenase, transfers

electrons from the substrate to FADH2 and then to

molecular O2 to yield H2O2 instead of capturing high-

energy electrons as FADH2 for use in the electron-

transport chain, as in mitochondrial β oxidation.

229
230
• This strong and potentially damaging oxidant is
immediately cleaved to H2O and O2 by catalase
which is abundantly present in the
peroxisomes

• In peroxisomes, the energy released in the first

oxidative step of fatty acid breakdown is not
conserved as ATP, but is dissipated as heat

231
2. Second difference is that peroxisomal thiolase
prefers longer chain fatty acids and has little
activity on acyl-CoAs with chains shorter than 8
carbons and that is why the peroxisomal β-
oxidation sequence ends at octanoyl-CoA (8 C).

• Octanoyl and acetyl groups are both further

oxidized in mitochondria

232
• The inability to oxidize these compounds is
responsible for several serious human diseases.

• Individuals with Zellweger syndrome are unable to

make peroxisomes and therefore lack all the
metabolism unique to that organelle.

• In X-linked adrenoleukodystrophy (XALD),

peroxisomes fail to oxidize very-long-chain fatty
acids, apparently for lack of a functional transporter
for these fatty acids in the peroxisomal membrane.
233
• Both defects lead to accumulation in the blood of
very-long-chain fatty acids, especially cerotic acid
(26:0).

• The exact cause for the varied collection of

symptoms found in the different ALD is not clear.

• The white matter of the brain, the Leydig cells of the

testes and the adrenal cortex are the most severely
affected systems.

234
• XALD affects young boys before the age of 10
years, causing behavioral disturbances, and death
within a few years

• Untreated, cerebral ALD is characterized by

progressive demyelination leading to a vegetative
state and death

• Patients of Zellweger syndrome usually do not

survive beyond one year of age

235
 Branched-Chain Fatty Acids

• Two of the most common

branched-chain fatty acids in

the diet are phytanic acid

(20C) and pristanic acid

(19C), which are degradation

products of chlorophyll, and

thus are consumed in green

vegetables
Phytanic Acid
236
• Animals do not synthesize branched-chain fatty
acids

• Branched-chain, 20 carbon fatty acid, phytanic

acid is not a substrate for acyl CoA
dehydrogenase because of the methyl group on its
β carbon

• These branched fatty acids are catabolized in

peroxisomes of animal cells by α oxidation

237
α-oxidation Pathway

1. Activation of Phytanic Acid

• Phytanic acid is first activated to phytanoyl CoA by

acyl CoA synthetase enzyme

238
2. Hydroxylation

• Phytanoyl CoA is hydroxylated on its α carbon, in a

reaction that involves molecular oxygen.

Dioxygenases

239
3. Decarboxylation

• It is then decarboxylated to form an aldehyde one

carbon shorter (Pristanal=19C)

Formyl group

240
4. Oxidation

• It is then oxidized to
the corresponding
carboxylic acid
which now has no
substituent on the β
carbon and can be
oxidized further by
β-oxidation

241
• Notice that α-oxidation of pristanic acid

releases propionyl-CoA, not acetyl-CoA

which is then converted to succinyl CoA to

enter into the citric acid cycle (End product

of 20 C phytanic acid)

242
Refsum’s disease is caused by a deficiency in a
single peroxisomal enzyme, the phytanoyl CoA
hydroxylase that carries out α-oxidation of
phytanic acid

• Symptoms include retinitis pigmentosa and

polyneuropathy

• Because phytanic acid is obtained solely from the

diet, placing patients on a low–phytanic acid diet
results in marked improvement
243
Summary: In the oxidation of phytanic acid,
for example, phytanoyl-CoA is:
i. Hydroxylated on its α carbon, in a reaction
that involves molecular oxygen
ii. Decarboxylated to form an aldehyde one
carbon shorter
iii. Then oxidized to the corresponding
carboxylic acid, which now has no
substituent on the β carbon and can be
oxidized further by β oxidation
244
The ω-Oxidation of Fatty Acids occurs in the
Endoplasmic Reticulum

• Although mitochondrial β oxidation, in which

enzymes act at the carboxyl end of a fatty acid, is
by far the most important catabolic fate for fatty
acids in animal cells, there is another pathway in
some species, including vertebrates, that involves
oxidation of the ω (omega) carbon

245
• The enzymes unique to ω oxidation are located in
the endoplasmic reticulum of liver and kidney, and
the preferred substrates are fatty acids of 10 or 12
carbon atoms (medium chain fatty acids)

• In mammals ω oxidation is normally a minor

pathway for fatty acid degradation, but when β
oxidation is defective (because of mutation or a
carnitine deficiency, for example) it becomes more
important
246
 Oxidases Use Oxygen As A Hydrogen Acceptor

• Oxidase is the general name for enzymes that catalyze

oxidations in which molecular oxygen is the electron acceptor
but oxygen atoms do not appear in the oxidized product

• Oxidases catalyze the removal of hydrogen from a substrate

using oxygen as a hydrogen acceptor

• They form water or hydrogen peroxide as a reaction product

247
 Oxygenases

• These enzymes catalyze

the incorporation of
molecular oxygen into the
substrate

• They may be
monooxygenses or
dioxygenases that
incorporate one or more
atoms of molecular oxygen
(O₂)
248
• Dioxygenases catalyze reactions in which both oxygen
atoms of O2 are incorporated into the organic substrate
molecule.

• The majority of dioxygenases fully incorporate dioxygen into

a single substrate, and a variety of cofactor schemes are
utilized to achieve this.

• For example, in the α-ketoglutarate-dependent enzymes,

one atom of dioxygen is incorporated into two substrates,
with one always being α-ketoglutarate, and this reaction is
brought about by a mononuclear iron center

249
• Monooxygenases, catalyze reactions in which only
one of the two oxygen atoms of O2 is incorporated
into the organic substrate, the other being reduced
to H2O.

• Monooxygenases require two substrates to serve

as reductants of the two oxygen atoms of O2.

• The main substrate accepts one of the two oxygen

atoms, and a co-substrate furnishes hydrogen
atoms to reduce the other oxygen atom to H2O.
250
• The general reaction equation for monooxygenases is:

Where

AH is the main substrate and BH2 the cosubstrate.

• They are also sometimes called mixed-function

oxidases or mixed-function oxygenases, to indicate
that they oxidize two different substrates
simultaneously
251
• The first step of ω-oxidation introduces a
hydroxyl group onto the ω carbon.

• The oxygen for this group comes from molecular

oxygen (O2) in a complex reaction that involves
cytochrome P450 monooxygenase system and the
electron donor NADPH+H+

• Reactions of this type are catalyzed by mixed-

function oxidases (or monooxygenases)
252
H2O +

253
• Two more enzymes now act on the ω carbon:

i. Alcohol Dehydrogenase oxidizes the hydroxyl

group to an aldehyde

254
 ii. Aldehyde Dehydrogenase oxidizes the
aldehyde group to a carboxylic acid

• These reactions produce a fatty acid with a carboxyl

group at each end

255
• At this point, either end can be attached to
coenzyme A , and the molecule can enter the
mitochondrion and undergo β oxidation by the
normal route

• In each pass through the β -oxidation pathway, the

"double-ended" fatty acid yields dicarboxylic acids
such as succinic acid, which can enter the citric
acid cycle.

256
257
 Ketone Bodies

• In humans and most other mammals, acetyl-CoA formed

in the liver mitochondria during oxidation of fatty acids
can:

1. Enter the citric acid cycle

2. Take part in fatty acid synthesis (by moving out)

3. Undergo conversion to the “ketone bodies” which are:

i. Acetone

ii. Acetoacetate

iii. D-β-hydroxybutyrate; for export to other tissues

258
• The acetyl CoA formed in fatty acid oxidation enters
the citric acid cycle only if fat and carbohydrate
degradation are appropriately balanced.

• Acetyl CoA must combine with oxaloacetate to gain

entry to the citric acid cycle.

• The availability of oxaloacetate, however, depends on

an adequate supply of carbohydrates.

• Recall that oxaloacetate is normally formed from

pyruvate, the product of glucose degradation in
glycolysis.
259
260
• If carbohydrate is unavailable or improperly utilized,

the concentration of oxaloacetate is lowered and

acetyl CoA cannot enter the citric acid cycle.

 During a fast, the liver is flooded with fatty acids

mobilized from adipose tissue.

261
• The resulting elevated hepatic acetyl CoA produced

primarily by fatty acid degradation inhibits pyruvate

dehydrogenase, and activates pyruvate carboxylase.

• The OAA thus produced is used by the liver for

gluconeogenesis rather than for the TCA cycle

• In these conditions, Acetyl CoA is directed to produce

ketone bodies.

262
263
 Summary

• In fasting or diabetes, oxaloacetate is consumed to form

glucose by the gluconeogenic pathway and hence is

unavailable for condensation with acetyl CoA.

 Under these conditions, acetyl CoA is diverted to

the formation of ketone bodies (Ketogenesis)

• Abnormally high levels of ketone bodies are present in the

blood of untreated diabetics.

264
• The first step in the

formation of acetoacetate

(4C), occurring in the liver,

is the enzymatic

condensation of two

molecules of acetyl-CoA

(2C), catalyzed by thiolase

• This is simply the reversal

of the last step of β

oxidation
265
• Acetoacetyl-CoA, which is the starting material for
ketogenesis, also arises directly from the terminal four
carbons of a fatty acid during β-oxidation

266
• The acetoacetyl-CoA
(4C) then condenses
with acetyl-CoA (2C) to
form β-hydroxy-β-
methyl glutaryl-CoA
(HMG-CoA) (6C), by
the enzyme HMG-CoA
synthase

267
Glutaric Acid C₃H₆(COOH)₂

268
• β-hydroxy-β-
methylglutaryl-CoA
(HMG-CoA), is then
cleaved to free
acetoacetate and
acetyl-CoA by the
enzyme HMG-CoA
lyase

269
270
Both enzymes must be present in mitochondria
for ketogenesis to take place

 This occurs solely in liver

• Acetone is formed in very small amounts from

acetoacetate, which is easily decarboxylated, either
spontaneously or by the action of acetoacetate
decarboxylase

• Acetone, is non-metabolizable, and is exhaled

271
272
• The acetoacetate is reversibly reduced by D-β-
hydroxy butyrate dehydrogenase, a
mitochondrial enzyme, to D-β-hydroxybutyrate

• Equilibrium between Acetoacetate and β -

hydroxybutyrate is controlled by the mitochondrial
[NAD+]/[NADH] ratio, that is, the redox state.

• D-β-Hydroxybutyrate is quantitatively the

predominant ketone body present in the blood and
urine in ketosis
273
• Acetoacetate and D- β-hydroxybutyrate are

transported by the blood to extrahepatic

tissues, where they are converted to acetyl-

CoA and oxidized in the citric acid cycle,

providing much of the energy required by

tissues such as skeletal and heart muscle

and the renal cortex

274
• The brain, which preferentially uses glucose as
fuel, can adapt to the use of acetoacetate or D-β-
hydroxybutyrate under starvation and diabetes.

• In prolonged starvation, 75% of the fuel needs of

the brain are met by ketone bodies.

• The production and export of ketone bodies from

the liver to extrahepatic tissues allows continued
oxidation of fatty acids in the liver when acetyl-CoA
is not being oxidized in the citric acid cycle
275
• In the liver, much of the
acetyl CoA generated from
fatty acid oxidation is
converted to the ketone
bodies, acetoacetate and
β-hydroxybutyrate, which
enter the blood

• The liver synthesizes

ketone bodies but cannot
use them as a fuel

276
• In skeletal muscle
and other tissues,
these ketone bodies
are converted back to
acetyl CoA, which is
oxidized in the TCA
cycle with generation
of ATP

277
• Because individuals with untreated diabetes
produce large quantities of acetoacetate, their
blood contains significant amounts of
acetone, which is toxic

• Acetone is volatile and imparts a

characteristic “fruity” odor to the breath, which
is sometimes useful in diagnosing diabetes

278
• In extrahepatic tissues, D-β-hydroxybutyrate is
oxidized to acetoacetate by D-β-hydroxybutyrate
dehydrogenase

279
• The acetoacetate is activated to its coenzyme A ester by
transfer of CoA from succinyl-CoA, an intermediate of the
citric acid cycle, in a reaction catalyzed by β-ketoacyl-CoA
transferase, aka thiophorase aka succinyl CoA:
acetoacetate CoA transferase

280
281
• The acetoacetyl-CoA is then cleaved by thiolase to
yield two acetyl-CoAs, which enter the citric acid
cycle

282
283
• If the blood level is raised, oxidation of ketone
bodies increases until, at a concentration of ~12
mmol/L, the oxidative machinery is saturated.

• When this occurs, a large proportion of oxygen

consumption may be accounted for by the
oxidation of ketone bodies.

284
• Thus the ketone bodies are used as fuels in

all tissues except liver, which lacks

thiophorase

• The liver is therefore a producer of ketone

bodies for the other tissues, and not a

consumer
285
286
• The production and export of ketone bodies by the

liver allows continued oxidation of fatty acids with only

minimal oxidation of acetyl-CoA

• Moreover, the liver contains only a limited amount of

Coenzyme A, and when most of it is tied up in acetyl-

CoA, β oxidation slows for want of the free coenzyme

• The production and export of ketone bodies frees

coenzyme A, allowing continued fatty acid oxidation

287
Ketogenesis Is Regulated at Three Crucial
Steps

1. Ketosis does not occur in vivo unless there

is an increase in the level of circulating free
fatty acids that arise from lipolysis of
triacylglycerol in adipose tissue

• Free fatty acids are the precursors of

ketone bodies in the liver
288
289
• The liver, both in fed and in fasting conditions,
extracts about 30% of the free fatty acids passing
through it, so that at high concentrations the flux
passing into the liver is substantial

• Therefore, the factors regulating mobilization of

free fatty acids from adipose tissue are
important in controlling ketogenesis

290
291
2. After uptake by the liver, free fatty acids are
either β-oxidized to CO2 or ketone bodies
or esterified to triacylglycerol and
phospholipid

• There is regulation of entry of fatty acids into

the oxidative pathway by carnitine
acyltransferase-I (CAT-I)

292
• CAT-I activity is low in the fed state, leading
to depression of fatty acid oxidation, and
high in starvation, allowing fatty acid
oxidation to increase

• Malonyl-CoA, the initial intermediate in fatty

acid biosynthesis formed by acetyl-CoA
carboxylase in the fed state, is a potent
inhibitor of CAT-I
293
• Under normal conditions, free fatty acids

enter the liver cell in low concentrations and

nearly all are esterified to acylglycerols and

transported out of the liver in very low

density lipoproteins (VLDL)

294
• However, as the concentration of free fatty acids
increases with the onset of starvation, acetyl-CoA
carboxylase is inhibited directly by acyl-CoA, and
malonyl-CoA decreases, releasing the inhibition of
CAT-I and allowing more acyl-CoA to be β–oxidized

• Thus, β-oxidation from free fatty acids is

controlled by the CAT-I gateway into the
mitochondria

295
• The enzyme undergoes
allosteric activation by
citrate, which causes dimers
to polymerize

• The enzyme can be

allosterically inactivated by
long-chain fatty acyl CoA
(the end product of the
pathway), which causes its
depolymerization

296
297
3. In turn, the acetyl-CoA formed in β -oxidation is
oxidized in the citric acid cycle, or it enters the
pathway of ketogenesis to form ketone bodies

• As the level of serum free fatty acids is raised,

proportionately more free fatty acid is converted to
ketone bodies and less is oxidized via the citric acid
cycle to CO2

298
299
• Thus complete oxidation of 1 mol of palmitate involves a
net production of 106 mol of ATP via β -oxidation and
CO2 production in the citric acid cycle, whereas only 26
mol of ATP are produced when acetoacetate is the end
product and only 21 mol when β-hydroxybutyrate is the
end product

• Thus, ketogenesis may be regarded as a mechanism

that allows the liver to oxidize increasing quantities of
fatty acids within the constraints of a tightly coupled
system of oxidative phosphorylation
300
Ketone Bodies Are Overproduced in Diabetes
And during Starvation

• Starvation and untreated diabetes mellitus lead to

overproduction of ketone bodies, with several
associated medical problems

• During starvation, gluconeogenesis depletes citric

acid cycle intermediates, diverting acetyl-CoA to
ketone body production

301
• Conditions that promote
gluconeogenesis slow
the citric acid cycle and
enhance the conversion
of acetyl-CoA to
acetoacetate

• The released CoA allows

continued β oxidation of
fatty acids

302
• The resulting accumulation of acetyl-CoA
accelerates the formation of ketone bodies beyond
the capacity of extrahepatic tissues to oxidize
them

• The increased blood levels of acetoacetate and D-

β-hydroxybutyrate lower the blood pH, causing the
condition known as acidosis (Ketoacidosis)

303
• The carboxyl group of a ketone body has a

pKa of about 4

• Therefore, each ketone body loses a proton

[H+] as it circulates in the blood, which

lowers the pH of the body

304
• Therefore, the increased number of

circulating [H+] can cause severe acidosis

(ketoacidosis)

• Extreme acidosis can lead to coma and in

some cases death

305
306
• Ketone bodies in the blood and urine of
individuals with untreated diabetes can reach
extraordinary levels
–A blood concentration of 90 mg/l00 ml
(compared with a normal level of <3 mg/100 ml)
– Urinary excretion of 5,000 mg/24 hr (compared
with a normal rate of <125 mgl24 hr)

• This condition is called ketosis

307
• Individuals on very low-calorie diets, using the fats
stored in adipose tissue as their major energy
source, also have increased levels of ketone
bodies in their blood and urine

• These levels must be monitored to avoid the

dangers of acidosis and ketosis (ketoacidosis)

308
Formation, utilization, and excretion of ketone bodies

309
Biosynthesis Of Cholesterol

• Cholesterol is present in tissues and in plasma

either as free cholesterol or combined with a long-

chain fatty acid as cholesteryl ester, the storage

form.

• Cholesterol is the precursor of all other steroids in

the body, including corticosteroids, sex

hormones, bile acids, and vitamin D

310
311
Acetyl-CoA Is the Source of all Carbon

atoms in Cholesterol

• Cholesterol is a 27-carbon compound consisting

of 4 rings and a side chain.

• It is synthesized from acetyl CoA by a lengthy

pathway that may be divided into five stages

312
 Cholesterol is Made from Acetyl-CoA in Five Stages

1. Condensation of three acetyl CoA units to form a six-

carbon intermediate, mevalonate (6C)

2. Conversion of mevalonate to activated isoprene units

(5C)

3. Polymerization of six 5-carbon isoprene units to form

the 30-carbon linear sequalene (30 C)

4. Cyclization of sequalene to form the parent steroid

lanosterol (30C),

5. Formation of cholesterol (27C) from lanosterol

313
• The isoprene units that are the essential
intermediates in the pathway from acetate to
cholesterol are also precursors to many
other natural lipids

2 Methyl 1,3
Butadiene (C5H8)
314
Stage 1- Synthesis of Mevalonate from Acetyl
CoA
• The first stage in cholesterol biosynthesis leads to
the intermediate mevalonate
• Two molecules of acetyl-CoA condense to form
acetoacetyl-CoA
• The acetoacetyl-CoA, then condenses with a third
molecule of acetyl-CoA to yield the six-carbon
compound β-hydroxy-β methylglutaryl-CoA
(HMG-CoA)
315
316
• These first two reactions are catalyzed by thiolase

and HMG-CoA synthase, respectively

• The cytosolic HMG-CoA synthase in this pathway

is distinct from the mitochondrial isozyme that

catalyzes HMG-CoA synthesis in ketone body

formation

317
• The third reaction is the committed, regulatory
and rate-limiting step

• Reduction of HMG-CoA to mevalonate, for which

each of two molecules of NADPH donates two
electrons (total 4 electrons)

• HMG-CoA reductase, an integral membrane

protein of the smooth ER, is the major point of
regulation on the pathway to cholesterol

318
319
• This last step is the principal regulatory step in the
pathway of cholesterol synthesis and is the site of action of
the most effective class of cholesterol-lowering drugs, the
statins, which are HMG-CoA reductase inhibitors

320
321
Stage 2: Conversion of Mevalonate
to Two Activated Isoprenes

• In the next stage of cholesterol

synthesis, three phosphate groups are
sequentially transferred from three ATP
molecules to mevalonate

322
323
324
• The phosphate attached to the C-3 hydroxyl group
of mevalonate in the intermediate 3-phospho-5-
pyrophosphomevalonate is a good leaving group

• In the next step, both this phosphate and the

nearby carboxyl group leave, producing a double
bond in the five-carbon product, 3-isopentenyl
pyrophosphate (IPP)

• This is the first of the two activated isoprenes

central to cholesterol formation
325
• Isomerization of 3-isopentenyl pyrophosphate (IPP) yields
the second activated isoprene, 3,3- dimethylallyl
pyrophosphate (DPP)

326
H2C=CH2 (Ethylene)

H2C=CH (Vinyl)

H2C=CH-CH2- R (Allyl)

327
328
 Stage 3: Condensation of Six Activated Isoprene
Units to Form Squalene (30C)

• Squalene is synthesized from isopentenyl

pyrophosphate by the reaction sequence
C5 C10 C15 C30

• Isopentenyl pyrophosphate and dimethylallyl

pyrophosphate now undergo a head-to-tail
condensation, in which one pyrophosphate group is
displaced and a l0-carbon chain, geranyl
pyrophosphate (GPP), is formed
329
• Geranyl pyrophosphate undergoes another

head-to-tail condensation with isopentenyl

pyrophosphate, yielding the l5-carbon

intermediate farnesyl pyrophosphate (15C)

(FPP)

330
Condensation of Six Activated Isoprene Units
to Form Squalene

331
• Finally, two
molecules of
farnesyl
pyrophosphate
join head to head,
with the
elimination of both
pyrophosphate
groups, to form
squalene (30C)
332
• Initially, inorganic pyrophosphate is eliminated,
forming presqualene diphosphate, which is then
reduced by NADPH with elimination of a further
inorganic pyrophosphate molecule.

• Squalene is formed from six isoprenoid units

• Because three ATP are hydrolysed per mevalonate

residue converted to activated isoprene unit, a total
of eighteen ATPs are required to make the
polyisoprenoid squalene (linear compound)
333
334
Stage 4: Conversion of Squalene to the Four-
Ring Steroid Nucleus (Parent steroid-
Lanosterol)

• When the squalene molecule is represented, the

relationship of its linear structure to the cyclic
structure of the sterols becomes apparent

• All sterols have the four fused rings that form the
steroid nucleus, and all are alcohols, with a
hydroxyl group at C-3
335
336
• The action of squalene
monooxygenase adds
one oxygen atom from O2
to the end of the squalene
chain, forming an epoxide

• This enzyme is another

mixed-function oxidase

• NADPH reduces the other

oxygen atom of O2 to H2O

337
Ether

Epoxide

338
• The methyl group on C14 is transferred to C13
and that on C8 to C14 as cyclization occurs,
catalyzed by oxidosqualene-lanosterol
cyclase.

• Hydroxylation involving the third carbon

results in an alcoholic group at Carbon no 3.

339
340
341
• The double bonds of the
product, squalene 2,3-
epoxide, are positioned so
that a remarkable concerted
reaction can convert the
linear squalene epoxide to a
cyclic structure lanosterol,
which contains the four rings
characteristic of the steroid
nucleus

342
 Stage 5—Formation of Cholesterol (27C)

• The formation of cholesterol from lanosterol takes place

in the membranes of the endoplasmic reticulum and
involves changes in the steroid nucleus and the side
chain.

• Lanosterol (30C) is converted into cholesterol (27C) in a

multistep process by:

i. Removal of three methyl groups,

ii. The migration of one double bond, and

iii. The reduction of the other double bond by NADPH

343
 C24

C4 C14
C8

30C

27C
344
Cholesterol

345
• The methyl groups on C14 (One group) and C4
(two groups) are removed to form 14-desmethyl
lanosterol (29 C) and then zymosterol (27C).

• The double bond at C8 -C9 is subsequently moved

to C5 -C6 in two steps, forming desmosterol.

• Finally, the double bond of the side chain is

reduced by NADPH, producing cholesterol

346
30C
29C 27C

347
348
Regulation of Cholesterol Synthesis

1. Allosteric regulation

2. Transcriptional control

3. Translational control

4. Proteolytic degradation (post translational control)

5. Covalent modification

349
 Regulation of Cholesterol Synthesis

1. Allosteric Regulation

• Regulation of cholesterol synthesis is exerted near the

beginning of the pathway, at the HMG-CoA reductase
step.

• However, it is only hepatic synthesis that is inhibited by

dietary cholesterol.

• HMG-CoA reductase in liver is inhibited by

mevalonate, the immediate product of the reaction,
and by cholesterol, the main product of the pathway
350
351
2. Transcriptional Control (DNAmRNA)

• The rate of synthesis of reductase mRNA is

controlled by the sterol regulatory element binding
protein (SREBP).

• This transcription factor binds to a short DNA

sequence called the sterol regulatory element
(SRE) on the 5’ side of the reductase gene.

 It binds to the SRE when cholesterol levels are low

and enhances transcription.
352
• In its inactive state, the SREBP resides in the
endoplasmic reticulum membrane, where it is
associated with the SREBP cleavage activating
protein (SCAP), an integral membrane protein

SCAP is the cholesterol sensor.

• When cholesterol levels fall, SCAP escorts SREBP

in small membrane vesicles to the Golgi complex,
where it is released from the membrane by two
specific proteolytic cleavages.
353
Site 1 protease Site 2 protease
S1P S2P
354
• The first cleavage frees a fragment of SREBP from
SCAP, whereas the second cleavage releases the
regulatory DNA binding domain from the
membrane.

• The released protein migrates to the nucleus and

binds the SRE of the HMG-CoA reductase gene to
enhance its transcription.

355
• When cholesterol levels rise,

i. The proteolytic release of the SREBP is

blocked, and

ii. The SREBP in the nucleus is rapidly degraded.

• These two events halt the transcription of genes of

the cholesterol biosynthetic pathways

 A diurnal variation occurs both in cholesterol

synthesis and reductase activity

356
357
• What is the molecular mechanism that retains SCAP–
SREBP in the ER when cholesterol is present but allows
movement to the Golgi complex when cholesterol
concentration is low?

• When cholesterol is low, SCAP binds to vesicular proteins

that facilitate the transport of SCAP–SREBP to the Golgi
apparatus, as described before.

• When cholesterol is present, SCAP binds cholesterol, which

causes a structural change in SCAP so that it binds to
another endoplasmic reticulum protein called Insig (insulin
induced gene)
358
• Insig is the anchor that retains SCAP and thus
SREBP in the endoplasmic reticulum in the
presence of cholesterol.

• Thus, two distinct steroid–protein interactions serve

to prevent the inappropriate movement of SCAP–
SREBP to the Golgi complex.

359
360
3. Translational Control

• The rate of translation of HMG CoA

reductase mRNA is inhibited by metabolites

derived from mevalonate

361
4. Proteolytic Degradation of HMG-CoA reductase

• The degradation of the reductase is stringently

controlled.

• The enzyme is bipartite: its cytoplasmic domain carries

out catalysis and its membrane domain senses signals
that lead to its degradation.

• The membrane domain may undergo structural

changes in response to increasing concentrations of
sterols such as lanosterol.

362
• Under these conditions, the reductase appears to
bind to another subset of Insigs that are also
associated with the ubiquitinating enzymes.

• The reductase is polyubiquitinated and

subsequently extracted from the membrane.

• The extracted reductase is then degraded by the

proteasome.

363
364
• The combined regulation at the levels of

transcription, translation, and degradation

can alter the amount of enzyme in the cell

more than 200-fold.

365
5. Regulation By Covalent Modification

• In addition to the inductive and repressive influences

cited above, the activity of the reductase is also
regulated by phosphorylation and dephosphorylation

• Elevated glucagon levels increase phosphorylation of

the enzyme, thereby inactivating it, whereas
hyperinsulinemia increases the activity of the
reductase by activating phosphatases, which
dephosphorylate the reductase
366
367
• The enzyme that phosphorylates HMG-CoA

reductase is the AMP-activated protein

kinase (AMPK), which itself is regulated by

phosphorylation by the AMP-activated

protein kinase kinase (AMPKK)

368
• Thus, cholesterol synthesis decreases when
ATP levels are low and increases when ATP
levels are high

• The statin drugs, including simvastatin & lovastatin

are structural analogs of HMG CoA, and are
reversible, competitive inhibitors of HMG CoA
reductase

• They are used to decrease plasma cholesterol

levels in patients with hypercholesterolemia
369
370