Voet D., Voet Ju.G. Biochemistry

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where x is at least 5 and where (CH

)

can contain one or

more double bonds. The (CH

)

portion of the substrate is

always saturated. Double bonds are inserted between ex-

isting double bonds in the (CH

)

portion of the substrate

and the CoA group such that the new double bond is three

carbon atoms closer to the CoA group than the next double

bond (not conjugated to an existing double bond) and, in

animals, never at positions beyond C9. Mammalian termi-

nal desaturases are components of mini-electron-transport

systems that contain two other proteins: cytochrome b

and

NADH–cytochrome b

reductase. The electron-transfer

reactions mediated by these complexes occur at the inner

surface of the ER membrane (Fig. 25-41) and are therefore

not associated with oxidative phosphorylation.

a. Some Unsaturated Fatty Acids Must

Be Obtained in the Diet

A variety of unsaturated fatty acids may be synthesized

by combinations of elongation and desaturation reactions.

However, since palmitic acid is the shortest available fatty

acid in animals, the above rules preclude the formation of

the 

double bond of linoleic acid [

9,12

-octadecadienoic

acid; 18:2n–6 (this nomenclature is explained in Table

12-1)], a required precursor of prostaglandins. Linoleic

acid must consequently be obtained in the diet (ultimately

from plants that have ⌬

- and ⌬

-desaturases; it is abun-

dant in most vegetable oils) and is therefore termed an essen-

tial fatty acid. Indeed, animals maintained on a fat-free

diet develop an ultimately fatal condition that is initially

characterized by poor growth, poor wound healing, and

dermatitis. Linoleic acid is also an important constituent of

epidermal sphingolipids that function as the skin’s water-

permeability barrier.

Because of the inability of animal desaturases to add

double bonds to positions beyond C9, another essential

fatty acid is -linolenic acid [ALA; 

9,12,15

-octadecatrienoic

acid (18:3n–3, an –3 fatty acid)]. This fatty acid is a pre-

cursor to EPA (

5,8,11,14,17

-eicosapentaenoic acid; 20:5n–3)

and DHA (

4,7,10,13,16,19

-docosahexaenoic acid; 22:6n–3),

polyunsaturated –3 fatty acids recently found to be im-

portant dietary constituents (present in fish oils) that im-

prove cognitive function and vision, and contribute to pro-

tection against inflammation and cardiovascular disease.

DHA is, among other things, the predominant fatty acid in

the phospholipids of retinal rod outer segments. Substitu-

tion of DHA with otherwise identical –6 fatty acids in

phospholipids results in impaired visual acuity. Deficiency

of –3 polyunsaturated fatty acids in brain phospholipids is

associated with memory loss and diminished cognitive

function.

F. Synthesis of Triacylglycerols

Triacylglycerols are synthesized from fatty acyl-CoA esters

and glycerol-3-phosphate or dihydroxyacetone phosphate

(Fig. 25-42). The initial step in this process is catalyzed

either by glycerol-3-phosphate acyltransferase in mito-

chondria and the ER, or by dihydroxyacetone phosphate

acyltransferase in the ER or peroxisomes.In the latter case,

the product acyl-dihydroxyacetone phosphate is reduced

to the corresponding lysophosphatidic acid by an NADPH-

dependent reductase. The lysophosphatidic acid is con-

verted to a triacylglycerol by the successive actions of 1-

acylglycerol-3-phosphate acyltransferase, phosphatidic

acid phosphatase, and diacylglycerol acyltransferase. The

intermediate phosphatidic acid and 1,2-diacylglycerol

(DAG) can also be converted to phospholipids by the path-

ways described in Section 25-8. The acyltransferases are

not completely specific for particular fatty acyl-CoAs,

either in chain length or in degree of unsaturation, but in

triacylglycerols of human adipose tissue, palmitate tends to

be concentrated at position 1 and oleate at position 2.

a. Glyceroneogenesis Is Important for

Triacylglycerol Biosynthesis

The dihydroxyacetone phosphate used to make

glycerol-3-phosphate for triacylglycerol synthesis comes

either from glucose via the glycolytic pathway (Fig. 17-3) or

membrane-bound, nonheme iron–containing enzymes

catalyze the general reaction

(CH

)

(CH

)

SCoA NADH

(CH

)

(CH

)

SCoA

NAD

Section 25-4. Fatty Acid Biosynthesis 971

Figure 25-41 The electron-transfer reactions mediated by the



-fatty acyl-CoA desaturase complex. Its three proteins, desat-

urase, cytochrome b

, and NADH–cytochrome b

reductase, are



FAD



FADH

NADH–cytochrome

reductase

Stearoyl-CoA + 2H

+ O

desaturase

red

2Fe

NAD

NADH + H

desaturase

2Fe

(18:0-CoA)

Oleoyl-CoA

+ 2H

(

-18:1-CoA)

2cyt b

(ox)

2cyt b

(red)

situated in the endoplasmic reticulum membrane. [After Jeffcoat,

R., Essays Biochem. 15, 19 (1979).]

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 971

972 Chapter 25. Lipid Metabolism

–

C O

Dihydroxyacetone

phosphate

–

C O

Acyl-dihydroxyacetone

phosphate

C SCoAR H SCoA

dihydroxyacetone phosphate

acyltransferase

–

C H

Glycerol-3-

phosphate

–

Lysophosphatidic acid

C SCoAR H SCoA

glycerol-3-phosphate

acyltransferase

HHO

NADP

NADPH

+ H

acyl-dihydroxyacetone

phosphate reductase

NAD

NADH + H

glycerol-3-phosphate

dehydrogenase

1-acylglycerol-3-phosphate

acyltransferase

C SCoAR

SCoA

–

Phosphatidic acid

CR

phosphatidic acid

phosphatase

1,2-Diacylglycerol (DAG)

CR

diacylglycerol

acyltransferase

C SCoAR

SCoA

Triacylglycerol

R

CR

C R

C SCoAR H SCoA

2-monoacylglycerol

acyltransferase

2-Monoacylglycerol

(from intestinal digestion)

CR

Phospholipids

Figure 25-42 The reactions of triacylglycerol biosynthesis.

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 972

from oxaloacetate via an abbreviated version of gluconeo-

genesis (Fig. 23-8) termed glyceroneogenesis. Glyceroneo-

genesis is necessary in times of starvation, since approxi-

mately 30% of the fatty acids that enter the liver during a

fast are reesterified to triacylglycerol and exported as

VLDL (Section 25-1 and 25-6A).Adipocytes also carry out

glyceroneogenesis in times of starvation.They do not carry

out gluconeogenesis but contain the gluconeogenic en-

zyme phosphoenolpyruvate carboxykinase (PEPCK),

which is upregulated when glucose concentration is low,

and participates in the glyceroneogenesis required for tria-

cylglycerol biosynthesis.

5 REGULATION OF FATTY ACID

METABOLISM

Discussions of metabolic control are usually concerned

with the regulation of metabolite flow through a pathway

in response to the differing energy needs and dietary states

of an organism. For example, the difference in the energy

requirement of muscle between rest and vigorous exertion

may be as much as 100-fold. Such varying demands may be

placed on the body when it is in either a fed or a fasted

state. For instance, Eric Newsholme, an authority on the

biochemistry of exercise, enjoys a 2-hour run before break-

fast. Others might wish for no greater exertion than the

motion of hand to mouth. In both individuals, glycogen and

triacylglycerols serve as primary fuels for energy-requiring

processes and are synthesized in times of quiet plenty for

future use.

a. Hormones Regulate Fatty Acid Metabolism

Synthesis and breakdown of glycogen and triacylglyc-

erols, as detailed in Chapter 18 and above, are processes that

concern the whole organism, with its organs and tissues

forming an interdependent network connected by the blood-

stream. The blood carries the metabolites responsible for

energy production: triacylglycerols in the form of chylomi-

crons and VLDL (Section 12-5A), fatty acids as their albu-

min complexes (Section 25-1e), ketone bodies, amino acids,

lactate, and glucose. The pancreatic  and  cells sense the

organism’s dietary and energetic state mainly through the

glucose concentration in the blood. The  cells respond to

the low blood glucose concentration of the fasting and

energy-demanding states by secreting glucagon.The  cells

respond to the high blood glucose concentration of the fed

and resting states by secreting insulin. We have previously

discussed (Sections 18-3E and 18-3F) how these hormones

are involved in glycogen metabolism. They also regulate the

rates of the opposing pathways of lipid metabolism and

therefore control whether fatty acids will be oxidized or syn-

thesized. Their targets are the regulatory (flux-generating)

enzymes of fatty acid synthesis and breakdown in specific

tissues (Fig. 25-43).

We are already familiar with most of the mechanisms by

which the catalytic activities of regulatory enzymes may be

controlled: substrate availability, allosteric interactions,

and covalent modification (phosphorylation). These are

examples of short-term regulation, regulation that occurs

with a response time of minutes or less. Fatty acid synthesis

is controlled, in part, by short-term regulation. Acetyl-CoA

carboxylase, which catalyzes the first committed step of

this pathway, is inhibited by palmitoyl-CoA and by the

glucagon-stimulated cAMP-dependent increase in phos-

phorylation, and is activated by citrate and by insulin-

stimulated dephosphorylation (Section 25-4B).

Another mechanism exists for controlling a pathway’s

regulatory enzymes: alteration of the amount of enzyme

present by changes in the rates of protein synthesis and/or

breakdown. This process requires hours or days and is

therefore called long-term regulation (the control of pro-

tein synthesis and breakdown is discussed in Chapters 31

and 32). Lipid biosynthesis is also controlled by long-term

regulation, with insulin stimulating and starvation inhibit-

ing the synthesis of acetyl-CoA carboxylase and fatty acid

synthase. The presence in the diet of polyunsaturated fatty

acids also decreases the concentrations of these enzymes.

The amount of adipose tissue lipoprotein lipase, the en-

zyme that initiates the entry of lipoprotein-packaged fatty

acids into adipose tissue for storage (Section 12-5Ba), is

also increased by insulin and decreased by starvation. In

contrast, the concentration of heart lipoprotein lipase,

which controls the entry of fatty acids from lipoproteins

into heart tissue for oxidation rather than storage, is de-

creased by insulin and increased by starvation. Starvation

and/or regular exercise, by decreasing the glucose concentra-

tion in the blood, change the body’s hormone balance. This

situation results in long-term changes in gene expression that

increase the levels of fatty acid oxidation enzymes and de-

crease those of lipid biosynthesis.

Fatty acid oxidation is regulated largely by the concen-

tration of fatty acids in the blood, which is, in turn, con-

trolled by the hydrolysis rate of triacylglycerols in adipose

tissue by hormone-sensitive triacylglycerol lipase. This

enzyme is so named because it is susceptible to regulation

by phosphorylation and dephosphorylation in response to

hormonally controlled cAMP levels. Epinephrine and

norepinephrine, as does glucagon, act to increase adipose

tissue cAMP concentrations. cAMP allosterically acti-

vates protein kinase A (PKA) which, in turn, increases

the phosphorylation levels of susceptible enzymes. Phos-

phorylation activates hormone-sensitive triacylglycerol li-

pase, thereby stimulating lipolysis in adipose tissue, raising

blood fatty acid levels, and ultimately activating the -

oxidation pathway in other tissues such as liver and muscle.

In liver, this process leads to the production of ketone

bodies that are secreted into the bloodstream for use by

peripheral tissues as an alternative fuel to glucose. PKA,

acting in concert with AMP-dependent protein kinase

(AMPK), also causes the inactivation of acetyl-CoA car-

boxylase (Section 25-4B), one of the rate-determining en-

zymes of fatty acid synthesis, so that cAMP-dependent

phosphorylation simultaneously stimulates fatty acid oxida-

tion and inhibits fatty acid synthesis.

Section 25-5. Regulation of Fatty Acid Metabolism 973

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 973

Insulin has the opposite effect of glucagon and epineph-

rine: It stimulates the formation of glycogen and triacyl-

glycerols. This protein hormone, which is secreted in re-

sponse to high blood glucose concentrations, triggers a

highly complex signal transduction network (Section 19-4F)

that induces the long-term regulation of numerous enzymes

as well as decreasing cAMP levels. This latter situation

leads to the dephosphorylation and thus the inactivation of

hormone-sensitive triacylglycerol lipase, thereby reducing

the amount of fatty acid available for oxidation. Insulin

974 Chapter 25. Lipid Metabolism

Mitochondrion

Citric acid

cycle

Citrate

Ketone

bodies

Acetyl-CoA

Fatty acyl-CoA

Fatty acid

Citrate

Acetyl-CoA

Malonyl-CoA

Palmitate

Stearate

Oleate

Smooth

fatty acid

synthase

Triacylglycerol

Hepatocyte (liver cell)

Acetyl-CoA

carboxylase

Ketone

bodies

Fatty acid oxidationFatty acid biosynthesis

Triacylglycerol

transport

Fatty acid

transport

Inhibited by

cAMP-dependent

phosphorylation

Activated by

insulin-dependent

dephosphorylation

Free fatty

acids

Triacylglycerols

Activated by

cAMP-dependent

phosphorylation

Fatty acid release

from adipocytes

into bloodstream

Very low density

lipoprotein (VLDL)

Fat storage

Adipocyte

"hormone-

sensitive"

lipase

Under long-term

Inhibition

Activation

regulation

Fatty acid

albumin complex

Lipoprotein

lipase

Figure 25-43 Sites of regulation of fatty acid metabolism.

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 974

also stimulates the dephosphorylation of acetyl-CoA car-

boxylase, thereby activating this enzyme (Section 25-4Ba).

The glucagon–insulin ratio is therefore of prime impor-

tance in determining the rate and direction of fatty acid

metabolism.

Another control point that inhibits fatty acid oxida-

tion when fatty acid synthesis is stimulated is the inhibi-

tion of carnitine palmitoyltransferase I by malonyl-CoA.

This inhibition keeps the newly synthesized fatty acids

out of the mitochondrion (Section 25-2B) and thus away

from the -oxidation system. As we have seen (Section

25-4Bc), heart muscle, an oxidative tissue that does not

carry out fatty acid biosynthesis, contains an isoform of

acetyl-CoA carboxylase, ACC2, whose sole function ap-

pears to be the synthesis of malonyl-CoA to regulate

fatty acid oxidation.

AMPK may itself be an important regulator of fatty

acid metabolism.This phosphorylating enzyme is activated

by AMP and inhibited by ATP and thus has been proposed

to serve as a fuel gauge for the cell. When ATP levels are

high, signaling the fed and rested state, this kinase is inhib-

ited, allowing ACC to become dephosphorylated (acti-

vated) so as to stimulate malonyl-CoA production for fatty

acid synthesis in adipose tissue and for inhibition of fatty

acid oxidation in muscle cells.When activity levels increase

causing ATP levels to decrease with a concomitant increase

in AMP levels, AMPK is activated to phosphorylate (inac-

tivate) ACC. The resulting decrease in malonyl-CoA levels

causes fatty acid biosynthesis to decrease in adipose tissue

while fatty acid oxidation increases in muscle to provide

the ATP for continued activity.

6 CHOLESTEROL METABOLISM

Cholesterol is a vital constituent of cell membranes and the

precursor of steroid hormones and bile salts. It is clearly es-

sential to life, yet its deposition in arteries has been associ-

ated with cardiovascular disease and stroke, two leading

causes of death in humans. In a healthy organism, an intri-

cate balance is maintained between the biosynthesis, utiliza-

tion, and transport of cholesterol, keeping its harmful depo-

sition to a minimum. In this section, we study the pathways

of cholesterol biosynthesis and transport and how they are

controlled. We also examine how cholesterol is utilized in

the biosynthesis of steroid hormones and bile salts.

A. Cholesterol Biosynthesis

All of the carbon atoms of cholesterol are derived from

acetate (Fig. 25-44). Observation of their pattern of incor-

poration into cholesterol led Konrad Bloch to propose that

acetate was first converted to isoprene units, C

units that

have the carbon skeleton of isoprene:

Isoprene units are condensed to form a linear precursor to

cholesterol, and then cyclized.

Squalene, a polyisoprenoid hydrocarbon (Fig. 25-45a),

was demonstrated to be the linear intermediate in choles-

terol biosynthesis by the observation that feeding isotopi-

cally labeled squalene to animals yields labeled choles-

terol. Squalene may be folded in several ways that would

enable it to cyclize to the four-ring sterol nucleus (Section

12-1E).The folding pattern proposed by Bloch and Robert

B.Woodward (Fig. 25-45b) proved to be correct.

Isoprene

(2-Methyl-1,3-butadiene)

An isoprene unit

CHC

CCC C

Section 25-6. Cholesterol Metabolism 975

Figure 25-44 All of cholesterol’s carbon atoms are derived

from acetate.

Acetate

–

CCC

(a)

(b)

Figure 25-45 Squalene. (a) Extended conformation. Each box contains one isoprene

unit. (b) Folded in preparation for cyclization as predicted by Bloch and Woodward.

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 975

Bloch’s outline for the major stages of cholesterol

biosynthesis was

This pathway has been experimentally verified and its de-

tails elaborated. It is now known to be part of a branched

pathway (Fig. 25-46) that produces several other essential

isoprenoids in addition to cholesterol, namely, ubiquinone

(CoQ; Fig. 22-17b), dolichol (Fig. 23-15), farnesylated and

geranylgeranylated proteins (Fig. 12-29), and isopentenyl-

adenosine (a modified base of tRNA; Fig. 32-10). We shall

examine in detail the portion of this pathway that synthe-

sizes cholesterol. Note, however, that 25,000 isoprenoids

(also known as terpenoids), mostly of plant, fungal, and

bacterial origin, have been characterized. These serve as

membrane constituents (e.g., cholesterol), hormones

squalene

cyclization product

cholesterol

Acetate

isoprenoid intermediate

(steroids), pheromones, defensive agents, photoprotective

agents (e.g., -carotene; Section 24-2Ad), and visual pig-

ments (e.g., retinal; Section 12-3Ab), to name only a few of

their many biological functions.

a. HMG-CoA Is a Key Cholesterol Precursor

Acetyl-CoA is converted to isoprene units by a series of

reactions that begins with formation of hydroxymethylglu-

taryl-CoA (HMG-CoA; Fig. 25-26), a compound we previ-

ously encountered as an intermediate in ketone body

biosynthesis (Section 25-3). HMG-CoA synthesis requires

the participation of two enzymes: thiolase and HMG-CoA

synthase. The enzymes forming the HMG-CoA leading to

ketone bodies occur in the mitochondria, whereas those re-

sponsible for the synthesis of the HMG-CoA that is des-

tined for cholesterol biosynthesis are located in the cytosol.

Their catalytic mechanisms, however, are identical.

976 Chapter 25. Lipid Metabolism

Figure 25-46 The branched pathway of isoprenoid

metabolism in mammalian cells. The pathway produces

ubiquinone, dolichol, farnesylated and geranylgeranylated

cis -Prenyl

transferase

Acetyl-CoA

HMG-CoA

Mevalonate

pyrophosphate

Isopentenyl

pyrophosphate

Dimethylallyl

pyrophosphate

Isopentenyl

adenosine

(tRNA)

Geranyl pyrophosphate

Farnesyl

pyrophosphate

Squalene

synthase

Squalene

CholesterolUbiquinone Dolichol

HMG-CoA reductase

trans -Prenyl

transferase

Geranyl-

geranyl

phosphate

Geranyl-

geranylated

proteins

Protein

prenyl

transferase

Farnesylated

proteins

proteins, and isopentenyl adenosine, a modified tRNA base, in

addition to cholesterol.

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 976

HMG-CoA is the precursor of two isoprenoid interme-

diates, isopentenyl pyrophosphate and dimethylallyl

pyrophosphate:

The formation of isopentenyl pyrophosphate involves four

reactions (Fig. 25-47):

1. The CoA thioester group of HMG-CoA is reduced

to an alcohol in an NADPH-dependent four-electron re-

duction catalyzed by HMG-CoA reductase, yielding

mevalonate.

2. The new OH group is phosphorylated by mevalonate-

5-phosphotransferase.

3. The phosphate group is converted to a pyrophos-

phate by phosphomevalonate kinase.

4. The molecule is decarboxylated and the resulting alco-

hol dehydrated by pyrophosphomevalonate decarboxylase.

HMG-CoA reductase mediates the rate-determining step

of cholesterol biosynthesis and is the most elaborately regu-

lated enzyme of this pathway. This 888-residue ER

membrane-bound enzyme is regulated, as we shall see in

Section 25-6Bb, by competitive and allosteric mechanisms,

phosphorylation/dephosphorylation, and long-term regu-

lation.Cholesterol itself is an important feedback regulator

of the enzyme.

b. Pyrophosphomevalonate Decarboxylase

Catalyzes an Apparently Concerted Reaction

5-Pyrophosphomevalonate is converted to isopentenyl

pyrophosphate by an ATP-dependent dehydration–decar-

boxylation reaction catalyzed by pyrophosphomevalonate

decarboxylase (Fig. 25-48). When [3-

O]5-pyrophospho-

mevalonate (*O in Fig. 25-48) is used as a substrate, the la-

beled oxygen appears in P

. This observation suggests that

3-phospho-5-pyrophosphomevalonate is a reaction inter-

mediate. Since all attempts to isolate this intermediate

have failed, however, it has been proposed that phosphory-

lation, the , elimination of CO

, and the elimination of P

occur in a concerted reaction.

OOP

–

Isopentenyl pyrophosphate

OOP

–

Dimethylallyl pyrophosphate

Section 25-6. Cholesterol Metabolism 977

Figure 25-47 Formation of isopentenyl pyrophosphate from HMG-CoA.

C SCoA

–

HMG-CoA

–

Mevalonate

mevalonate-5-

phosphotransferase

ATP

ADP

Phosphomevalonate

–

phosphomevalonate

kinase

ATP

ADP

5-Pyrophosphomevalonate

–

pyrophospho-

mevalonate

decarboxylase

ATP

ADP

+ P

+ CO

Isopentenyl pyrophosphate

–

HMG-CoA

reductase

CoA

2 NADP

2 NADPH

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 977

–

Isopentenyl

pyrophosphate

Cys

–

Tertiary

carbocation

–

Cys

Glu

–

Glu

–

Dimethylallyl

pyrophosphate

–

Cys

Glu

The equilibration between isopentenyl pyrophosphate

and dimethylallyl pyrophosphate is catalyzed by isopen-

tenyl pyrophosphate isomerase. The reaction appears to

occur via a protonation/deprotonation reaction involving

the intermediacy of a tertiary carbocation intermediate.

Cys and Glu residues have been implicated as the general

acid and base catalysts, respectively (Fig. 25-49), as sup-

ported by site-directed mutagenesis and the X-ray struc-

ture of the enzyme. The carbocation is thought to be sta-

bilized through interactions with the aromatic  cloud of

an adjacent Trp residue. Aromatic residues provide elec-

tron-rich interactions with positively charged groups

without forming covalent bonds that would destroy the

intermediate.

c. Squalene Is Formed by the Condensation

of Six Isoprene Units

Four isopentenyl pyrophosphates and two dimethylallyl

pyrophosphates condense to form the C

cholesterol pre-

cursor squalene in three reactions catalyzed by two en-

zymes (Fig. 25-50):

1. Prenyltransferase (farnesyl pyrophosphate synthase)

catalyzes the head-to-tail (1¿–4) condensation of dimethy-

lallyl pyrophosphate and isopentenyl pyrophosphate to

yield geranyl pyrophosphate.

2. Prenyltransferase catalyzes a second head-to-tail

condensation of geranyl pyrophosphate and isopentenyl

pyrophosphate to yield farnesyl pyrophosphate (FPP).

3. Squalene synthase (SQS) then catalyzes the head-to-

head (1–1¿) condensation of two farnesyl pyrophosphate

molecules to form squalene. Farnesyl pyrophosphate is

also a precursor to dolichol, farnesylated and geranylger-

anylated proteins, and ubiquinone (Fig. 25-46).

Prenyltransferase catalyzes the condensation of isopen-

tenyl pyrophosphate with an allylic (conjugated to a C“C

double bond) pyrophosphate. It is specific for isopentenyl

pyrophosphate but can use either the 5-carbon dimethyl-

allyl pyrophosphate or the 10-carbon geranyl pyrophosphate

as its allylic substrate.The prenyltransferase-catalyzed con-

densation mechanism is particularly interesting since it is

one of the few known enzyme-catalyzed reactions that pro-

978 Chapter 25. Lipid Metabolism

Figure 25-48 Action of pyrophosphomevalonate decarboxylase. The enzyme catalyzes an ATP-dependent

concerted dehydration–decarboxylation of pyrophosphomevalonate, yielding isopentenyl pyrophosphate.

Figure 25-49 Mechanism of isopentenyl pyrophosphate

isomerase. The enzyme interconverts isopentenyl pyrophosphate

and dimethylallyl pyrophosphate by a protonation/deprotonation

reaction involving a carbocation intermediate in which a Cys and

a Glu residue act as a proton donor and acceptor.The carbo-

cation intermediate appears to be stabilized by  interactions

with a nearby Trp side chain.

OOP

–

ADP

OOP

–

+ ADP +

–

pyrophospho-

mevalonate

decarboxylase

5-Pyrophosphomevalonate Isopentenyl pyrophosphate

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 978

ceed via a carbocation intermediate. Two possible conden-

sation mechanisms can be envisioned (Fig. 25-51):

Scheme I An S

1 mechanism in which an allylic carbo-

cation forms by the elimination of PP

. Isopentenyl py-

rophosphate then condenses with this carbocation, forming

a new carbocation that eliminates a proton to form product.

Scheme II An S

2 reaction in which the allylic PP

displaced in a concerted manner. In this case, an enzyme

nucleophile, X, assists in the reaction. This group is elimi-

nated in the second step with the loss of a proton to form

product.

Dale Poulter and Hans Rilling used chemical logic to

differentiate between these two mechanisms. Capitalizing

on the observation that S

1 reactions are much more sensi-

tive to electron-withdrawing groups than S

2 reactions,

they synthesized a geranyl pyrophosphate derivative in

which the H at C2 is replaced by the electron-withdrawing

group F. This allylic substrate for the second (1¿–4) conden-

sation catalyzed by prenyltransferase, not surprisingly, has

the same K

as the natural substrate (F and H have similar

atomic radii):

It is, however,the V

max

of this reaction that tells the story. If

the reaction is an S

2 displacement, the fluoro derivative

OPP

, H

OPP

Section 25-6. Cholesterol Metabolism 979

Figure 25-50 Formation of squalene from isopentenyl

pyrophosphate and dimethylallyl pyrophosphate. The

pathway involves two head-to-tail condensations catalyzed

by prenyltransferase and a head-to-head condensation

catalyzed by squalene synthase.

Squalene

squalene synthase

(head to head)

NADPH

NADP

+ 2 PP

prenyltransferase

(head to tail)

prenyltransferase

(head to tail)

P PO

Farnesyl pyrophosphate

Geranyl pyrophosphate

Dimethylallyl pyrophosphate

Isopentenyl pyrophosphate

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 979

should react at a rate similar to that of the natural sub-

strate. If, instead, the reaction has an S

1 mechanism, the

fluoro derivative should react orders of magnitude more

slowly than the natural substrate. In fact, 3-fluorogeranyl

pyrophosphate forms product at 1% of the rate of the

natural substrate, strongly supporting an S

1 mechanism

with a carbocation intermediate.

Carbocations are now known to participate in several

reactions of isoprenoid biosynthesis. The enzymes are clas-

sified according to how they generate these carbocations.

Class I enzymes do so via the release of pyrophosphate, as

we have seen for prenyltransferase. Class II enzymes do so

by protonating a double bond, as does isopentenyl py-

rophosphate isomerase (Fig. 25-49), or an epoxide, as we

shall see below for oxidosqualene cyclase.

Squalene, the immediate sterol precursor, is formed by

the head-to-head condensation of two FPP molecules by

SQS.Although the enzyme is a Class I enzyme that is struc-

turally related to prenyltransferase and generates carboca-

tions by the release of pyrophosphate, the reaction is not a

simple head-to-tail condensation, as might be expected,

but, rather, proceeds via a complex two-step mechanism

with each step catalyzed by a different active site on the en-

zyme (Fig. 25-52):

Step I The reaction of two FPP molecules to yield the

stable intermediate presqualene pyrophosphate. This reac-

980 Chapter 25. Lipid Metabolism

OP P

Scheme I

Ionization–condensation–elimination

OP P

P P

OP P

Scheme II

Condensation–elimination

P P

–

1

NADP

+ PP

NADPH

OPP

Farnesyl

pyrophosphate

Farnesyl

pyrophosphate

Presqualene

pyrophosphate

Squalene

Figure 25-51 Two possible mechanisms for

the prenyltransferase reaction. Scheme I

involves the formation of a carbocation

intermediate, whereas Scheme II involves the

participation of an enzyme nucleophile, X.

Figure 25-52 Action of squalene synthase. The enzyme

catalyzes the head-to-head condensation of two farnesyl

pyrophosphate molecules to form squalene.

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