33a Fatty Acid Release 33b Fatty Acid Transport 33c Fatty Acid Oxidation 33d Ketone Bodies III METABOLIC BIOCHEMISTRY 33a Fatty Acid Release Lipolysis ID: 907960
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Slide1
§3.3 Lipid Catabolism §3.3a Fatty Acid Release §3.3b Fatty Acid Transport §3.3c Fatty Acid Oxidation §3.3d Ketone Bodies
III. METABOLIC BIOCHEMISTRY
Slide2§3.3a Fatty Acid Release (Lipolysis)
Slide3Synopsis 3.3a
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty
Acid
Release (luminal/cytosolic)
—
the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)
Slide4Coenzyme A: A common metabolic cofactorCoenzyme A (CoA) is involved in numerous metabolic pathways, including: (1)
B
iosynthesis of fatty acids
(2) Oxidation of fatty acids
(3) Oxidation of pyruvate
Slide5Fatty Acids: A Few Common Members
O
-
10
O
O
-
12
O
O
-
14
O
O
-
16
O
Structure
Acid
[
HA
]
Base/Salt
[
A
-
]
x
:m
Acyl Group
[R—C(O)—]
12:0
Lauric
Acid
Laurate
Lauroyl
14:0
Myristic
Acid
Myristate
Myristoyl
16:0
Palmitic Acid
Palmitate
Palmitoyl
18:0
Stearic Acid
Stearate
Stearoyl
Slide6
c
is-
9-dodecanoate
While the
x:m symbolism
provides insights into the length and the degree of unsaturation of a fatty acid (see
§1.3
)
, an
alternative nomenclature
is needed to indicate both the
position
and the stereochemistry of the double bond(s)
In this nomenclature, the position and
stereochemical
configuration
of C=C double bond is indicated by
the
z-
n
notation
:
=> unsaturation within the C=C bond
z
=> cis/trans stereochemistry about the C=C bond
n => numeric
position of
first
C atom within C=C bond from carboxyl end
For example, the cis-
9 notation is indicative of a C=C double bond beginning @ C9 within the fatty acid
tail harboring cis-configuration
What does
trans-2 suggest?!
Fatty Acids: Nomenclature
Slide7Lipase
Fatty Acids
-
O
-
O
-O
+
Triglyceride Breakdown
Triglycerides (or
triacylglycerols
)
are fatty acid esters (usually with different fatty acid R groups) of glycerol—see §1.3!
The fats in the diet are
largely t
riglycerides—they are also
stored in the adipose tissue where they function as “
high-energy
” reservoirs
In order to release such energy to be used as “
free energy
”, triglycerides are first de-esterified or hydrolyzed into free fatty acids by lipases via a process known as “
lipolysis
”
Dietary triglycerides
are broken down in the lumen of small intestine (through the action of pancreatic lipase)—the resulting fatty acids and glycerol are absorbed into blood through the intestinal microvilli and transported to
hepatocytes in the liver
In adipose tissue
, the breakdown of triglycerides occurs within the cytosol of adipocytes (through the action of hormone-sensitive lipase expressed by adipocytes)—the resulting fatty
acids and glycerol
are secreted into the blood for transport to
hepatocytes in the liver
Slide8Exercise 3.3a
Describe
the chemical structure of CoA
What is the functional group in CoA that participates in covalent linkage with fatty acids
In the context of fatty acid nomenclature,
what does the
trans-7 notation imply?How are fatty acids released from their parent fats?
Slide9§3.3b Fatty Acid Transport
Slide10Synopsis 3.3b
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty
Acid
Release (luminal/cytosolic)
—
the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)
Slide11(A) FA Transport: OverviewPrior to their oxidation within the mitochondria, the fatty acids are first imported from the cytosolSuch import requires the
“priming” of
fatty
acids with
coenzyme A (CoA)
so as to
generate the acyl-CoA derivative within the cytosol—eg lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA, and stearoyl-CoA (12:0, 14:0, 16:0, and 18:0) Recall that acyl is a functional group with the general formula R-C=O, where R is an alkyl sidechain (or in this case, the non-polar tail of fatty acids)Given the rather charged character of CoA moiety (vide infra), acyl-CoA produced in the cytosol cannot cross (or diffuse through) the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix (the site of Krebs cycle)Accordingly, acyl-CoA is subjected to reversible conversion to acyl-carnitine in order to exploit the carnitine shuttle system located within the IMM to translocate it to the mitochondrial matrix Acyl-CoA synthetase
Fatty Acid
1
2
3
4
Acyl-CoA (cytosolic)
Acyl-CoA (mitochondrial matrix)
Acyl-carnitine
Acyl-carnitine
Carnitine
acyltransferase
I
Carnitine-
acylcarnitine
translocase
(Mitochondrial Transit)
Carnitine
acyltransferase
II
Slide12FA Transport: (1) Acyl-CoA Synthetase
In order to be oxidized to provide free energy, fatty acids are first “primed” with CoA in an ATP-dependent reaction to generate the
acyl-CoA derivative within the cytosol
The reaction is catalyzed
by a family of enzymes called “
acyl-CoA
synthetases” or “thiokinases”First step mediated via nucleophilic attack of O atom of fatty acid carboxylate anion on the -phosphate of ATP to generate the acyladenylate mixed anhydride intermediate and PPi—which undergoes exergonic hydrolysis to Pi to drive the reaction to completionSecond-step involves nucleophilic attack by the thiol (-SH) group of CoA on the carbonyl C atom
of
acyladenylate
mixed anhydride
intermediate to generate
acyl-COA
and
AMP
The overall result is that
the free energy of fatty acid is conserved via the generation of a “high-energy” thioester bond of acyl-CoA within the
cytosol
—but
how does acyl-CoA get
into
the mitochondrial matrix (the site of
Krebs cycle)?
Slide13FA Transport: (2) Carnitine Acyltransferase
I
Given the rather charged character of CoA moiety
, acyl-CoA produced in the cytosol cannot
cross (or diffuse through)
the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix (the site of
Krebs cycle)Accordingly, acyl-CoA is first converted to acyl-carnitine by carnitine acyltransferase I—an enzyme located at the outer (intermembraneous space) surface of IMM—in order to exploit the carnitine shuttle system for its delivery into the mitochondrial matrixCarnitine, a quaternary amine, has no known physiological function other than its role in the shuttling of fatty acids from the intermembraneous space to mitochondrial matrix Note that the free energy of thioester bond in acyl-CoA is conserved in the ester (or O-acyl) bond in acyl-carnitine Carnitine
Acyl-carnitine
Acyl-CoA
CoA
Carnitine
acyltransferase
I
Slide14FA Transport: (3) Carnitine-Acylcarnitine TranslocaseAcyl-carnitine is shuttled across the inner mitochondrial membrane (
IMM)—from the cytosol (or the
intermembraneous
space) to the mitochondrial matrix—by the
carnitine-
acylcarnitine
translocaseCarnitine-acylcarnitinetranslocaseAcyl-carnitine
Cytosol
(intermembrane
space)
Mitochondrial
Matrix
Acyl-carnitine
Slide15FA Transport: (4) Carnitine Acyltransferase II Inside the mitochondrial matrix, c
arnitine acyltransferase
II
catalyzes the reverse transfer of acyl group of acyl-carnitine back to CoA to generate acyl-CoA and free carnitine
Acyl-CoA
is then not only “chemically” but also “spatially” primed
to be converted to acetyl-CoA for subsequent entry into the Krebs cycleCarnitineAcyl-carnitine
Acyl-CoA
CoA
Carnitine
acyltransferase
II
Slide16FA Transport: OutlineAcyl-CoA is transported from the cytosol (or the intermembraneous space) to the mitochondrial matrix by the carnitine shuttle system
as follows:
Fatty acid is “primed” with CoA in the cytosol
Acyl group of cytosolic acyl-CoA is transferred to
carnitine
acyl-carnitine
Acyl-carnitine is shuttled across the IMM into the mitochondrial matrix by carnitine-acylcarnitine translocaseAcyl group of matrix acyl-carnitine is transferred to mitochondrial matrix CoA acyl-CoA, thereby freeing up free carnitine poolFree carnitine within the matrix is shuttled back to the cytosol to repeat the cycle Carnitine-acylcarnitinetranslocase2
3
5
4
Carnitine
acyltransferase I
Carnitine
acyltransferase II
RCO
OH
SCoA
1
Slide17Exercise 3.3b
Describe the activation of fatty acids. What is the energy cost for the process?
How
does carnitine shuttle transport fatty acids into the mitochondrial matrix?
Distinguish between the roles and subcellular localization of carnitine acyltransferases I and II?
Slide18§3.3c Fatty Acid Oxidation
Slide19Synopsis 3.3c
In the context of generating free energy, lipid catabolism can be subdivided into three stages:
Fatty
Acid
Release (luminal/cytosolic)
—
the breakdown of fats or triglycerides in the diet (in the lumen of small intestine after a meal) or adipose tissue (in the cytosol of adipocytes during starvation) into fatty acids by a group of enzymes collectively referred to as “lipases”—this process is technically called “lipolysis” Fatty Acid Transport (cytosolic)—In the cytosol, the released fatty acids are first activated via covalent linkage to coenzyme A (CoA) so as to generate acyl-CoA harboring a “high-energy” thioester bond and then subsequently transported into the mitochondrial matrix via a carnitine shuttle Fatty Acid Oxidation (mitochondrial)—In the mitochondrial matrix, fatty acids undergo multiple rounds of so-called “β-oxidation”, with each round producing reduced cofactors FADH2 and NADH (destined for electron transport chain) as well as acetyl-CoA (destined for Krebs cycle)
Slide20(B) FA Oxidation: Overview Within the mitochondrial matrix, oxidation of acyl-CoA into acetyl-CoA (a Krebs cycle
substrate) occurs via four distinct
steps
—each
requiring the involvement of a specific mitochondrial
enzyme
This process is referred to as “-oxidation”—due to the fact that the acyl group of acyl-CoA is oxidized at its -carbon atom in a repetitive fashion so as to degrade fatty acids with the removal of a two-carbon unit in the form of acetyl-CoA during each round A common mechanism to cleave the C—C bond involves the following four steps: Dehydrogenate: H2C—CH2 HC=CH Hydroxylate: HC=CH HC(OH)—CH2 Oxidize: HC(OH)—CH2 C(O)—CH2 Cleave via nucleophilic attack: C(O)—CH2 - Let us see that in action!
Acyl-CoA dehydrogenase
1
2
3
4
trans-
2
-Enoyl-CoA
Acetyl-CoA
-Ketoacyl-CoA
L-
-Hydroxyacyl-CoA
Enoyl-CoA hydratase
-Ketoacyl-CoA thiolase
Acyl-CoA
-Hydroxyacyl-CoA dehydrogenase
Slide21FA Oxidation: (1) Acyl-CoA Dehydrogenase
Dehydrogenation
Dehydrogenation of saturated C-C single bond within acyl-CoA results in the formation of
enoyl
-CoA harboring a C=C double bond
Since such dehydrogenation begins at C atom numbered 2, the product is prefixed with trans-2 to indicate the stereochemical configuration and position of the C=C double bond Reaction catalyzed by acyl-CoA dehydrogenase using FAD as an oxidizing agent (more powerful than NAD+) or electron acceptor—thus the energy released due to the oxidation of acyl group is conserved in the form of FADH2
FADH
2
will be subsequently
reoxidized
back to FAD via the mitochondrial
electron transport chain (ETC)
Slide22FA Oxidation: (2) Enoyl-CoA Hydratase
L-
-Hydroxyacyl-CoA
Hydration
Hydration
of unsaturated C=C double bond within trans-2-enoyl-CoA (prochiral) results in the formation of L--hydroxyacyl-CoAReaction catalyzed by enoyl-CoA hydratase in a stereospecific manner producing exclusively the L-isomerThe addition of an –OH group at the C position “primes” L--hydroxyacyl-CoA for subsequent oxidation to a keto group—the C atom of which then serves as an electrophilic center for the release of first acetyl-CoA
Slide23FA Oxidation: (3) -Hydroxyacyl-CoA Dehydrogenase
Oxidation
Oxidation of –OH to a
keto
group
at the C position within L--
hydroxyacyl-CoA results in the formation of corresponding -ketoacyl-CoAReaction catalyzed by -hydroxyacyl-CoA dehydrogenase using NAD+ as an oxidizing agent or electron acceptor—the energy of electron transfer is conserved in NADH NADH will be subsequently reoxidized back to NAD+ via the mitochondrial electron transport chain (ETC)
L-
-Hydroxyacyl-CoA
-hydroxyacyl-CoA dehydrogenase
Slide24ThiolysisThiolysis (or breaking bonds with –SH group—cf hydrolysis and phosphorolysis) initiated by nucleophilic attack of the thiol group (-SH) of CoA on the keto group within -
ketoacyl
-CoA results in the cleavage of C-C bond,
thereby releasing the first acetyl-CoA
(to enter the Krebs cycle) and
an outgoing acyl-CoA
Reaction catalyzed by -ketoacyl-CoA thiolase The outgoing acyl-CoA is two C atoms shorter than the parent acyl-CoA that entered the first round of -oxidation—this acyl-CoA will undergo subsequent rounds of -oxidation (Steps 1-4) to generate additional acetyl-CoA molecules—how many?!Complete -oxidation of a 2n:0 fatty acid requires n-1 rounds—ie it will generate n acetyl-CoA, n-1 NADH, and n-1 FADH2! That would be bucketloads of energy—but exactly how much?!
FA Oxidation: (4)
-
Ketoacyl
-CoA
Thiolase
Palmitoyl-CoA
8 Acetyl-CoA
7 NADH
7 FADH
2
8 FADH
2
24 NADH
8 GTP
10.5 ATP
17.5 ATP
60 ATP
12 ATP
8 ATP
Total Energy = 108 ATP
Krebs cycle
ETC
ETC
ETC
ETC
-
Oxidation
Palmitic
acid is a saturated fatty acid harboring 16 carbon atoms (16:0)
It is the most commonly occurring fatty acid in living organisms
So how much energy does -oxidation of a single chain of
palmitic
acid (16 C atoms) generate?
Complete degradation of
palmitic
acid would require 7 rounds of -oxidation producing
7 FADH
2
,
7 NADH
and
8 acetyl-CoA
—the final round produces 2 acetyl-CoA!
Further oxidation of each acetyl-CoA via the Krebs cycle produces
3 NADH
,
1 FADH
2
and
1 GTP
(enzymatically converted
to
ATP) per molecule (and there are 8 acetyl-CoA!)—see
§
3.5
Oxidation of each
NADH
and
FADH2
via the ETC respectively produces
2.5
and
1.5
molecules of
ATP
—see
§
3.6
6
Palmitic
Acid (16:0)
FA Oxidation:
Bucketloads
of ATP
Fat Is
hypercaloric
!
Slide26Exercise 3.3c
Summarize the chemical reactions that occur in each round of
β
-oxidation. Explain why
this
process is called
β-oxidation?How is ATP recovered from the products of β-oxidation?How many rounds of β-oxidation are needed to completely oxidize an 18:0 fatty acid? How many of the following are produced: acetyl-CoA, NADH, and FADH2?
Slide27§3.3d Ketone Bodies
Slide28Synopsis 3.3d
While
acetyl-CoA
produced via fatty acid oxidation is by and large
funneled into the Krebs cycle in most tissues, it can also be converted
to the so-called ketone bodies in a process referred to as “ketogenesis”—the conversion of ketone bodies back into acetyl-CoA is called “ketolysis” Ketone bodies—essentially acetyl-CoA-in-disguise—include small water-soluble molecules such as acetoacetate, -hydroxybutyrate , and acetoneKetogenesis primarily occurs within the mitochondrial matrix of liver cells under conditions of starvation during glucose shortage—the metabolic state under which the body derives some of its energy from the use of ketone bodies as metabolic fuels is called “ketosis”—eg the body being in a state of ketosis vs state of glycolysis Conditions such as alcohol consumption, ketogenic (fat-rich) diet, prolonged starvation, and diabetes mellitus can result in the production of ketone bodies in a rather high concentration in the blood—such metabolic state is referred to as “
ketoacidosis
”
Ketoacidosis
results in a decrease in blood pH and is fraught with serious pathological consequences—fruit-like smell of breath due to acetone may be a sign of ketoacidosis!
W
hy is there a need to produce
ketone bodies
?!!
Slide29Ketone Bodies: Physiological SignificanceBeing small and water-soluble, ketone bodies represent a neat trick to transport acetyl-CoA from liver to peripheral tissues (to be used as a metabolic fuel) such as the:
Heart (virtually no glycogen reserves)
—since heart primarily relies on fatty acids for energy production
, ketone bodies serve as an alternative source of fuel that can be readily “burned” via the Krebs cycle to generate energy
Brain (low glycogen reserves that likely mediate neuronal activity rather than glucose metabolism)
—since fatty acids and acetyl-CoA cannot enter the brain
due to the presence of the so-called blood-brain-barrier (BBB), the ability of ketone bodies to diffuse (via monocarboxylate transporters) through the BBB renders them perfect candidates as an alternative source of fuel (when glucose is in short supply) and as precursors for fatty acid biosynthesisBBB is an highly selective filter/barrier that separates the circulating blood in the brain from the extracellular fluid—only water, gases, and lipophilic molecules such as steroid hormones can usually cross the BBB by passive diffusion Typical CapillaryBrain Capillary
Slide30SCoA
Acetyl-CoA
Acetoacetate
(
-
Ketobutyrate
)Ketone Bodies: KetogenesisNADH
NAD
+
-
hydroxybutyrate
dehydrogenase
H
-
H
ydroxybutyrate
CO
2
Acetoacetate
d
ecarboxylase
(or spontaneously)
3
Acetone
-
hydroxybutyrate
is easily converted back to acetyl-CoA via acetoacetate
Conversion of acetone back to acetyl-CoA occurs via lactate and pyruvate in the liver
Ketone bodies include:
Acetoacetate
-
hydroxybutyrate
Acetone
How is acetyl-CoA converted to ketone bodies in the liver?
How are ketone bodies converted back to acetyl-CoA in target tissues so as to be utilized as a source of fuel via the Krebs cycle?
However, acetone is usually excreted via urine and/or exhaled
Slide31The conversion of acetyl-CoA to ketone bodies such as acetoacetate in the liver occurs via three major enzymatic steps (ketogenesis):Thiolase condenses two molecules of acetyl-CoA into acetoacetyl-CoA
Hydroxymethylglutaryl
-CoA synthase
adds another molecule of acetyl-CoA to
acetoacetyl
-CoA to generate -
hydroxy--methylglutaryl-CoAHydroxymethylglutaryl-CoA lyase breaks down -hydroxy--methylglutaryl-CoA into acetyl-CoA and acetoacetate—one of the three ketone bodies Ketone Bodies: (1) Acetyl-CoA Acetoacetate [Liver]Glutaric Acid (5C)
1
2
3
Slide321
2
3
Ketone bodies
such as acetoacetate and -
hydroxybutyrate
(produced by the liver) travel in the bloodstream to reach tissues such as the heart and brain, where they are converted back to acetyl-CoA
via the following enzymatic steps (
ketolysis
):
-
hydroxybutyrate
dehydrogenase
mediates the oxidation of -
hydroxybutyrate
into acetoacetate
Ketoacyl
-CoA transferase
condenses
acetoacetate
with CoA (donated by
succinyl
-CoA)
to generate
acetoacetyl
-CoA
Thiolase
breaks down
acetoacetyl
-CoA into two acetyl-CoA molecules using free CoA as a nucleophile
The newly generated
acetyl-CoA
can now
serve either as a
Krebs cycle
substrate
for
energy production (or
as a precursor for fatty acid biosynthesis!)
Ketone Bodies: (2)
Acetoacetate
Acetyl-CoA [
Heart|Brain]
Slide33Exercise 3.3d
What
are ketone bodies
?
Which organs utilize ketone bodies as an alternative source of fuel?
How
are ketone bodies synthesized and degraded?