Chapter 25 lipid synthesis

• 25.1 Fatty Acid Biosynthesis & Degradation • 25.2 Biosynthesis of Complex Lipids • 25.3 Eicosanoid Biosynthesis and Function • 25.4 Cholesterol Biosynthesis • 25.5 Transport via Lipopr

Chapter 25

Lipid Biosynthesis

• 25.1 Fatty Acid Biosynthesis & Degradation

• 25.2 Biosynthesis of Complex Lipids

• 25.3 Eicosanoid Biosynthesis and Function

• 25.4 Cholesterol Biosynthesis

• 25.5 Transport via Lipoprotein Complexes

• 25.6 Biosynthesis of Bile Acids

• 25.7 Synthesis and Metabolism of Steroids

Fatty Acid Pathways

The Biosynthesis and Degradation

Pathways are Different

• As in cases of glycolysis/gluconeogenesis and glycogen synthesis/breakdown, fatty acid synthesis and degradation go by

different routes

• There are 4 major differences between fatty acid breakdown and biosynthesis

• Enzymes of synthesis are one polypeptide

• Biosynthesis uses NADPH/NADP+;

(breakdown uses NADH/NAD+)

Acetate Unit Activation by

Malonyl-CoA for Fatty Acid Synthesis

Acetate Units are Activated for Transfer in Fatty

Acid Synthesis by Malonyl-CoA The design strategy for fatty acid synthesis is:

• Fatty acids are built from 2-C units: acetyl-CoA

• Acetate units are activated for transfer by

conversion to malonyl-CoA

• Chain grows to 16-carbons

• Other enzymes add double bonds and more Cs

Challenge: Ac-CoA in Cytosol

What are the sources?

Sufficient quantities of acetyl-CoA, malonyl-CoA, and

NADPH must be generated in the cytosol for f.a synthesis

• A.a degradation produces cytosolic acetyl-CoA

• F.a oxidation produces mitochondrial acetyl-CoA

• Glycolysis yields cytosolic pyruvate which is

converted to acetyl-CoA in mitochondria

• Citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents for fatty acid synthesis

• NADPH can be produced in the pentose phosphate

pathway

Figure 25.1 · The citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents (electrons) for f.a synthesis

The shuttle collects carbon substrates, primarily from glycolysis but also from f.a oxidation and a.a catabolism

Most of the reducing equivalents are glycolytic in origin

Pathways that provide carbon for f.a synthesis: in blue; pathways that supply electrons for f.a synthesis: in red

Acetyl-CoA Carboxylase (ACC)

The "ACC enzyme" commits acetate to fatty

acid synthesis

• Carboxylation of acetyl-CoA to form

malonyl-CoA is the irreversible, committed step in fatty acid biosynthesis (Fig 25.2)

• ACC uses bicarbonate and ATP (and biotin!)

• E.coli enzyme has three subunits

• Animal enzyme is one polypeptide with all three functions - biotin carboxyl carrier,

biotin carboxylase and transcarboxylase

Figure 25.2

( a ) The acetyl-CoA

carboxylase reaction produces malonyl-CoA for fatty acid synthesis ( b ) A mechanism for the acetyl-CoA

carboxylase reaction ( Step 1 ) Bicarbonate is activated for

carbonylphosphate

intermediate

( Step 2 ) In a typical biotin-dependent

reaction, nucleophilic attack by the acetyl- CoA carbanion on the carboxyl carbon of N- carboxybiotin - a

transcarboxylation

-yields the carboxylated product.

Acetyl-CoA Carboxylase II

ACC forms long, active filamentous

polymers from inactive protomers

• As a committed step, ACC is carefully

regulated

• Palmitoyl-CoA (product) favors

monomers

• Citrate favors the active polymeric form

• Phosphorylation modulates citrate

activation and palmitoyl-CoA inhibition

protein kinases responsible.

Phosphorylation

at Ser1200 is

primarily

responsible for decreasing the affinity for citrate.

The Effect of Phosphorylation

• Unphosphorylated E has low Km for

citrate and is active at low citrate

• Unphosphorylated E has high Ki for

palm-CoA and needs high palm-CoA to inhibit

• Phosphorylated E has high Km for citrate and needs high citrate to activate

• Phosphorylated E has low Ki for

palm-CoA and is inhibited at low palm-palm-CoA

The Acyl Carrier Protein

Carrier of intermediates

in fatty acid synthesis

• Acetyl and malonyl groups- the basic building blocks

of f.a synthesis- are not transferred directly from

CoA to the growing f.a chain

• They are first passed to acyl carrier protein (ACP)

• This protein consists (in E coli) of a single

polypeptide chain of 77 residues to which is

attached a phosphopante-theine group

(the same group forming the "business end" of CoA)

• Thus, in terms of function, ACP is a large CoA,

specialized for use in f.a biosynthesis

See Figure 25.6 to compare ACP and CoA

Figure 25.6 · Fatty acids are conjugated both to

coenzyme A and to acyl carrier protein (ACP) through the sulfhydryl of phosphopantetheine

prosthetic groups

Fatty Acid Synthesis in Bacteria

• Other three steps are VERY familiar!

• Only differences: D configuration and NADPH

• Check equations in textbooks!

Acetyl and malonyl

building blocks are

introduced as acyl carrier protein conjugates.

Decarboxylation drives the b -ketoacyl-ACP

synthase and results in the addition of 2-C units

to the growing chain

Concentrations of free f.a are extremely low in

most cells

Newly synthesized f.a

exist primarily as CoA esters.

acyl-Decarboxylation Drives the Condensation

of Acetyl-CoA and Malonyl-CoA

• The first actual elongation reaction involves the condensation of acetyl-ACP and malonyl-ACP by the b-ketoacyl-ACP synthase to form

acetoacetyl-ACP (Fig 25.7)

• One might ask at this point: Why is the 3-C

malonyl group used here as a 2-C donor?

• The answer is that this is yet another example

of a decarboxylation driving a

thermo-dynamically unfavorable reaction

• The decarboxylation that accompanies the

reaction with malonyl-ACP drives the synthesis

of acetoacetyl-ACP

• ATP is indirectly responsible for the

condensation reaction to form acetoacetyl-ACP

(as hydrolysis of ATP drove the carboxylation

of acetyl-CoA to form malonyl-ACP)

• Malonyl-CoA can be viewed as a form of stored energy for driving f.a synthesis

• All the C of acetoacetyl-ACP (and of the f.a to

be made) are derived from acetate units of

acetyl-CoA

(as carboxyl carbon that was added to drive

this reaction is the one removed by the

condensing enzyme)

Reduction of the b-Carbonyl Group

Follows a Now-Familiar Route

The next 3 steps look very similar to the f.a degradation pathway in reverse (Fig 25.7):

– reduction of the b -carbonyl group to form a b alcohol,

-– dehydration

– reduction to saturate the chain

but with 2 crucial differences :

1 the alcohol formed in the first step has the D configuration (rather than the L form seen in catabolism)

2 the reducing coenzyme is NADPH (NAD + and

• The net result of this biosynthetic cycle is the synthesis of a 4-C unit (a butyryl group)

from two smaller building blocks

• In the next cycle of the process, this ACP condenses with another malonyl-ACP to make a 6-C b-ketoacyl-ACP and CO2

butyryl-• Subsequent reduction to a b-alcohol,

dehydration, and another reduction yield a

6-C saturated acyl-ACP

• This cycle continues with the net addition of

a 2-C unit in each turn until the chain is 16 carbons long (Fig 25.7)

• The b-ketoacyl-ACP synthase cannot

accommodate larger substrates, so the reaction cycle ends with a 16-C chain

• Hydrolysis of the C16-acyl-ACP yields a palmitic acid and the free ACP

• Thus, 7 malonyl-CoA molecules and 1

acetyl-CoA yield a palmitate

(shown here as palmitoyl-CoA):

Acetyl-CoA + 7 malonyl-CoA 2 + 14 NADPH + 14 H + 

palmitoyl-CoA + 7 HCO 3 2 + 14 NADP+ + 7 CoASH

Fatty Acid Synthesis in Animals

Fatty Acid Synthase –

a multienzyme complex

• Dimer of 250 kD multifunctional polypeptides

• Note the roles of active site serines on AT & MT

• Study the mechanism in Figure 25.11 - note the roles of ACP and KSase

Figure 25.11 · The mechanism of the fatty acyl synthase reaction in eukaryotes

(1) Acetyl and malonyl groups are loaded onto acetyl transferase and malonyl transferase

(2) The acetate unit that forms the base of the nascent chain is transferred first to the acyl carrier protein domain and (3) then to the b -ketoacyl synthase

(4) Attack by ACP on the carbonyl C of a malonyl unit on malonyl transferase forms malonyl-ACP (5) Decarboxylation leaves a reactive, transient carbanion that can attack the carbonyl carbon of the acetyl group on the b-ketoacyl synthase.

(6) Reduction of the keto group, dehydration, and saturation of the resulting double bond follow,

leaving an acyl group on ACP, and steps 3 through 6 repeat to lengthen the nascent chain.

Further Processing of FAs

• Additional elongation - in mitochondria and ER

• Introduction of cis double bonds - do you need

O2 or not?

• E.coli add double bonds while the site of attack

is still near something functional (the thioester)

• Eukaryotes add double bond to middle of the chain - and need power of O2 to do it

• Polyunsaturated FAs - plants vs animals

Additional Elongation

• Palmitate is the primary product of the f.a synthase.

• Cells synthesize many other f.a

• Shorter chains are easily made if the chain is

released before reaching 16 C in length

• Longer chains are made through special elongation reactions, which occur both in the mitochondria and

at the surface of the endoplasmic reticulum (ER)

• The ER reactions are actually quite similar to those

we have just discussed

(addition of 2-C units at the carboxyl end of the

chain by oxidative decarboxylations involving

malonyl-CoA).

• This decarboxylation provides the thermodynamic driving force for the condensation reaction

• The mitochondrial reactions involve

addition (and subsequent reduction) of acetyl units

• These reactions (Figure 25.12) are

essentially a reversal of f.a oxidation, with the exception that NADPH is

utilized in the saturation of the double bond, instead of FADH2

Figure 25.12- Elongation of f.a in mitochondria is initiated by

the thiolase reaction

The b -ketoacyl intermediate thus formed undergoes the same three reactions (in

reverse order) that are the basis of b-oxidation of fatty acids Reduction of the b- keto

group is followed by dehydration to form a double bond Reduction of the double

bond yields a fatty acyl-CoA that is elongated by two carbons

Note that the reducing coenzyme for the second step is NADH, whereas the reductant

for the fourth step is NADPH

Introduction of a Single

cis Double Bond

Both prokaryotes and eukaryotes are capable of

introducing a single cis double bond in a newly

synthesized f.a There is a fundamental chemical difference between the two ways:

– Eukaryotes have adopted an O2-dependent

pathway: the reaction can occur anywhere in the f.a chain, with no (additional) need to activate the

desired bond toward dehydrogenation.

– Bacteria (such as E coli) carry out this process in

an O2-independent pathway: dehydrogenation

occurs while the bond of interest is still near the b carbonyl or b -hydroxy group and the thioester

-group at the end of the chain (as some other means must be found to activate the bond in question)

Figure 25.13

Double bonds are introduced into the growing fatty acid

chain in E coli by

specific dehydrases

Palmitoleoyl-ACP is synthesized by a sequence of

reactions involving

4 rounds of chain elongation, followed

by double bond

insertion by b

-hydroxydecanoyl thioester dehydrase and 3 additional

elongation steps.

Another elongation cycle produces cis- vaccenic acid

Biosynthesis of Polyunsaturated Fatty Acids

• Organisms differ with respect to formation,

processing, and utilization of polyunsaturated f.a

• Eukaryotes do synthesize a variety of

poly-unsaturated f.a

• Plants manufacture double bonds between the D 9

and the methyl end of the chain, but mammals

cannot

• Plants readily desaturate oleic acid at the

position (to give linoleic acid) or at both the

12-and 15-positions (producing linolenic acid)

• Mammals require polyunsaturated fatty acids, but must acquire them in their diet As such, they are

referred to as essential fatty acids

Arachidonic Acid Is Synthesized from

Linoleic Acid by Mammals

• Mammals can add additional double bonds to

unsaturated f.a in their diets

Example: their ability to make arachidonic acid (the precursor for biologically active derivatives such as prostaglandins, leukotrienes) from linoleic acid.

– A 2 nd desaturation reaction at the

C5-– Liberation of the product, a 20 C f.a with double bonds

at the 5-, 8-, 11-, 14-positions (20:4 ( D 5, 8, 11, 14 )

Regulation of FA Synthesis

Allosteric modifiers, phosphorylation and

hormones

• Malonyl-CoA blocks the carnitine

acyltransferase and thus inhibits b-oxidation

• Hormones regulate ACC

• Glucagon activates lipases/inhibits ACC

activity increases

• Rising citrate levels (which reflect an

abundance of CoA) similarly signal the initiation of f.a

acetyl-synthesis.

Hormonal Signals Regulate ACC

and Fatty Acid Biosynthesis

• Citrate activation and palmitoyl-CoA inhibition of acetyl-CoA carboxylase are strongly depen-dent

on the phosphorylation state of the enzyme

• This provides a crucial connection to hormonal regulation

• Many of the enzymes that act to phosphorylate acetyl-CoA carboxylase) are controlled by

hormonal signals

• Glucagon is a good example: is binding to

membrane receptors activates an intracellular

cascade involving activation of adenylyl cyclase

• Cyclic AMP produced by the cyclase activates a protein kinase, which then phosphorylates

acetyl-CoA carboxylase

• Unless citrate levels are high, phosphorylation causes inhibition of f.a biosynthesis

• The carboxylase (and f.a synthesis) can be

reactivated by a specific phosphatase, which dephosphorylates the carboxylase

• The simultaneous activation by glucagon of

triacylglycerol lipases, which hydrolyze

triacylglycerols, releasing f.a for b-oxidation

• Both the inactivation of acetyl-CoA

carboxylase and the activation of

triacylglycerol lipase are counteracted by

insulin, whose receptor acts to stimulate a

phosphodiesterase that converts cAMP to AMP

Figure 25.17

Hormonal

signals

regulate f.a synthesis,

depends upon hormonal

activation of triacylglycerol lipase.

• Complex lipids consist of backbone structures

to which fatty acids are covalently bound

• Principal classes include:

1 Glycerolipids, for which glycerol is the backbone,

having 2 major classes:

• Glycerophospholipids

• Triacylglycerols.

2 Sphingolipids, which are built on a sphingosine

backbone

• The phospholipids (include both

glycero-phospholipids and sphingomyelins) are:

– crucial components of membrane structure.

Biosynthesis of Complex Lipids

Synthetic pathways depend on organism

• Sphingolipids and triacylglycerols only made

in eukaryotes

• PE accounts for 75% of PLs in E.coli

• No PC, PI, sphingolipids, cholesterol in E.coli

• But some bacteria do produce PC

Glycerolipid Biosynthesis

CTP drives formation of CDP complexes

• Phosphatidic acid is the precursor for all other glycerolipids in eukaryotes

• See Fig.25.18

• PA is made either into DAG or CDP-DAG

• Note the roles of CDP-choline and ethanolamine in synthesis of PC and PE

CDP-in Fig 25.19

• Note exchange of ethanolamine for

serine (25.21)

Figure

25.18

Synthesis of glycerolipids

in

eukaryotes begins with the

formation of phosphatidic acid, which may be

formed from dihydroxy- acetone

phosphate

or glycerol

as shown