§ 3.3 Lipid Catabolism - PowerPoint Presentation

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)

Slide3

Synopsis 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)

Slide4

Coenzyme 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

Slide5

Fatty 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

Slide7

Lipase

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

Slide8

Exercise 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

Slide10

Synopsis 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

Slide12

FA 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)?

Slide13

FA 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

Slide14

FA 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

Slide15

FA 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

Slide16

FA 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

Slide17

Exercise 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

Slide19

Synopsis 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

Slide21

FA 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)

Slide22

FA 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

Slide23

FA 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

Slide24

ThiolysisThiolysis (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

Slide25

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

!

Slide26

Exercise 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

Slide28

Synopsis 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

?!!

Slide29

Ketone 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

Slide30

SCoA

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

Slide31

The 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

Slide32

1

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]

Slide33

Exercise 3.3d

What

are ketone bodies

?

Which organs utilize ketone bodies as an alternative source of fuel?

How

are ketone bodies synthesized and degraded?