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Cholesterol Synthesis

Copyright 1999-2008 by Joyce J. Diwan.

All rights reserved.

Molecular Biochemistry II

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Hydroxymethylglutaryl-coenzyme A (HMG-CoA)is the precursor for cholesterol synthesis.

HMG-CoA is also an intermediate on the pathway forsynthesis ofketone bodies from acetyl-CoA.

The enzymes for ketone body production are locatedin the mitochondrial matrix.

HMG-CoA destined for cholesterol synthesis is madeby equivalent, but different, enzymes in the cytosol.

CH2 C CH2 C

OH O

SCoA

CH3

C

O

O

hydroxymethylglutaryl- o

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HMG-CoA is formed by condensation of acetyl-CoA& acetoacetyl-CoA, catalyzed by HMG-CoA Synthase.

HMG-CoA Reductase catalyzes production of

mevalonate from HMG-CoA.

H3C C CH2 C

O O

SCoA

H3C C

O

SCoA

HSCoA

CH2 C CH2 C

OH O

SCoA

CH3

C

O

O

H2O acetoacetyl-CoA

hydroxymethylglutaryl-CoA

acetyl-CoA HMG-CoA

Synthase

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The carboxyl of HMG

that is in ester linkage tothe CoA thiol is reducedto an aldehyde, and thento an alcohol.

NADPH serves asreductant in the 2-stepreaction.

Mevaldehyde is thoughtto be an active siteintermediate, followingthe first reduction andrelease of CoA.

+ HSCoA

H2C

C

CH3

HO

CH2

C

O O

C SCoA

O

H2CC

CH3

HO

CH2

C

O O

H2C OH

2NADP+

2NADPH

HMG-CoA

mevalonate

HMG-CoAReductase

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HMG-CoA Reductase is an integral protein ofendoplasmic reticulum membranes.

The catalytic domain of this enzyme remains active

following cleavage from the transmembrane portionof the enzyme.

The HMG-CoA Reductase reaction, in whichmevalonate is formed from HMG-CoA, is rate-

limiting for cholesterol synthesis.

This enzyme is highly regulated and the target ofpharmaceutical intervention.

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Mevalonate is

phosphorylated by 2sequential Pi transfers

from ATP, yielding

the pyrophosphate

derivative.

ATP-dependent

decarboxylation, with

dehydration, yieldsisopentenyl

pyrophosphate.

H2CC

CH3HO

CH2

C

O O

CH2 OH

H2C

C

CH2 CH2 O P O P O

O

O

O

O

CH3

H2C

CCH3

HO

CH2C

O O

CH2 O P O P O

O

O

O

O

CO2

ATP

ADP Pi

2ATP

2ADP

mevalonate

5-pyrophosphomevalonate

(2 steps)

isopentenyl pyrophosphate

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Isopentenyl

pyrophosphate is

the first of several

compounds in thepathway that are

referred to as

isoprenoids, by

reference to thecompound isoprene.

isoprene

H2C

C

C

CH2

CH3

H

isopentenyl pyrophosphate

H2C

C

CH2

H2

C

CH3

O P

O

O

O P O

O

O

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Prenyl Transferase catalyzes head-to-tail condensations:

Dimethylallyl pyrophosphate & isopentenylpyrophosphate react to form geranyl pyrophosphate.

Condensation with another isopentenyl pyrophosphate

yields farnesyl pyrophosphate. Each condensation reaction is thought to involve a

reactive carbocation formed as PPi is eliminated.

Condensation Reactions

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CH2 CH2 O P O P O

O

O

O

O

CH CH2 O P O P O

O

O

O

O

CH2C

CH3

CH3C

CH3

CH CH2CH3C

CH3

CH CH2 O P O P O

O

O

O

O

CCH2

CH3

PPi

CH2 CH2 O P O P O

O

O

O

O

CH2C

CH3

CH CH2CH3C

CH3

CH CH2CCH2

CH3

PPi

CH CH2 O P O P O

O

O

O

O

CCH2

CH3

dimethylallyl pyrophosphate

isopentenyl pyrophosphate

isopentenyl pyrophosphate

geranyl pyrophosphate

arnesyl pyrophosphate

Each condensation involves a carbocation ormed as PPi is eliminated.

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Squalene Synthase: Head-to-head condensation of 2 farnesyl

pyrophosphate, with reduction by NADPH, yields squalene.

CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 O P O P O

O

O

O

O

CCH2

CH3

2

O

NADP+

O2 H2O

HO

H+

NADPH

NADP++ 2PPi

NADPH

2 farnesyl pyrophosphate

squalene 2,3-oxidosqualene lanosterol

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Squaline epoxidase catalyzes conversion ofsqualene to2,3-oxidosqualene.

This mixed function oxidation requires NADPH asreductant & O2 as oxidant. One O atom is incorporated into

substrate (as the epoxide) & the otherO is reduced to water.

O

NADP+

O2 H2O

HO

H+NADPH

squalene 2,3-oxidosqualene lanosterol

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Structural studies of a related bacterial enzyme have

confirmed that the substratebinds at the active site in aconformation that permits cyclization with only modestchanges in position as the reaction proceeds.

The product is the sterol lanosterol.

O HO

H+

2,3-oxidosqualene lanosterol

Squalene

Oxidocyclase

catalyzes a seriesof electron shifts,initiated by

protonation of the

epoxide, resultingin cyclization.

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Conversion oflanosterol to cholesterol involves 19

reactions, catalyzed by enzymes in ER membranes.Additional modifications yield the various steroidhormones orvitamin D.

HO HO

lanosterol cholesterol

19 steps

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Many of the reactions involved in converting lanosterolto cholesterol and other steroids are catalyzed bymembers of the cytochrome P450 enzyme superfamily.

The human genome encodes 57 members of the cyt P450superfamily, with tissue-specific expression andintracellular localization highly regulated.

Some P450 enzymes are localized in mitochondria.

Others are associated with endoplasmic reticulummembranes.

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Cyt P450 enzymes catalyze various oxidative reactions.

Many are mixed function oxidations (mono-oxygenations)that require O2 & a reductant,e.g.,NADPH.

One oxygen atom is incorporated into a substrate & theother oxygen atom is reduced to water.

An example is hydroxylation of a steroid as in the ERelectron transfer pathway above:

NADPH transfers 2 electrons to cytochrome P450 via a

reductase that has FAD & FMNprosthetic groups.

2e

NADPH FAD/FMN P450

ROH + H2O

RH + O2

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X

e

Y

A cysteine S atom typically serves as an axial ligand

(X or Y) for the iron atom of a cyt P450 heme.

The other axial position, where O2binds, may be open or

have a bound H2O that is displaced by O2.

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O2 is cleaved after binding to the reduced P450 heme iron.

In the example shown: one oxygen atom is reduced to water

and a substrate is hydroxylated.

2e

NADPH FAD/FMN P450

ROH + H2O

RH + O2

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Reactions catalyzed by different P450 enzymes includehydroxylation, epoxidation, dealkylation, peroxidation,deamination, desulfuration, dehalogenation, etc.

P450 substrates include steroids, polyunsaturated fattyacids, eicosanoids, retinoids, & various non-polar

xenobiotics (drugs & other foreign compounds).Some P450 enzymes have broad substrate specificity.

Mechanisms fordetoxification of non-polarcompounds include reactions such as hydroxylations

that increase polarity, so that the products of thesereactions can be excreted by the kidneys.

Explore with Chime the hemoprotein domain of aBacillusmagaterium cytochrome P450.

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CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 O P O P O

O

O

O

O

CCH2

CH3

farnesyl pyrophosphate

Farnesyl pyrophosphate, an intermediate on the pathwayfor cholesterol synthesis, also serves also as precursor forsynthesis of various non-steroidal isoprenoids.

The importance of the other products of the pathway thatoriginates with mevalonate is reflected in serious diseasesthat result from genetic defects in this pathway.

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CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 S Prot iCCH2

CH3

arnesyl residue linked to protein via cysteine

Farnesyl Transferase catalyzes transfer of the farnesylmoiety of farnesyl pyrophosphate to a cysteine residue in asequence CaaX at the C-terminus of a protein, "a" beingan aliphatic amino acid.

After subsequent cleavage of the terminal 3 amino acids,the new terminal carboxyl may be methylated, furtherincreasing hydrophobicity.

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Some otherisoprenoids:

Dolichol pyrophosphate has a role in synthesis ofoligosaccharide chains of glycoproteins.

Additional roles have been proposed; dolichol is foundin many membranes of cells.

H CH2 C CH CH2 CH2 CH CH2 CH2 O P O P O

CH3 CH3 O O

O

O16-19

olichol pyrophosphate

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Heme a, a constituent of respiratory chain complexes,

has a farnesyl side-chain.

N

N

N

N

CH3 HC

CH2

CH3

CH CH2

CH2

CH2

COO

CH3

HC

CH2CH2

OOC

Fe

OH

CH2 CH C CH2

CH3

3 H

O

Heme a

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Regulation of cholesterol synthesis

HMG-CoA Reductase, the rate-limiting step on thepathway for synthesis of cholesterol, is a major controlpoint. Regulation relating to cellular uptake ofcholesterol will be discussed in the next class.

Short-term regulation:HMG-CoA Reductase is inhibited by phosphorylation,catalyzed by AMP-Dependent Protein Kinase (whichalso regulates fatty acid synthesis and catabolism).

This kinase is active when cellular AMP is high,corresponding to when ATP is low.

Thus, when cellular ATP is low, energy is not expendedin synthesizing cholesterol.

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Long-term regulation is by varied formation and

degradation of HMG-CoA Reductase and other enzymesof the pathway for synthesis of cholesterol.

Regulated proteolysis ofHMG-CoA Reductase:

Degradation of HMG-CoA Reductase isstimulated by cholesterol, oxidized derivatives of

cholesterol, mevalonate, & farnesol

(dephosphorylated farnesyl pyrophosphate).

HMG-CoA Reductase includes a transmembrane

sterol-sensing domain that has a role in activating

degradation of the enzyme via the proteasome

(proteasome to be discussed later).

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Association with Insig causes the SREBP-SCAP

precursor complex to be retained within the ER.When sterol levels are low, SCAP & Insig do not interact.

This allows the SCAP-SREBP precursor complex totranslocate from the ER to the golgi apparatus.

SCAP has a transmembranesterol-sensing domain

homologous to that ofHMG-CoA Reductase.

When bound to a sterol, thesterol-sensing domain of

SCAP binds the ERmembrane protein Insig.

PreSREBP-SCAP/sterol-Insig

sterol

PreSREBP-SCAP-Insig

Insig

PreSREBP-SCAP(translocates to golgi)

(in ER)

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Protease S1P (site one

protease), an integralprotein of golgimembranes, cleaves theSREBP precursor at a

site in the lumenaldomain.

N

membrane

cytosol

golgilumen

S2P cleavagereleasing

SREBP

S P-activatedS1P cleavage

An intramembrane zinc metalloprotease domain of

another golgi protease S2P then catalyzes cleavagewithin the transmembrane segment of the SREBPprecursor, releasing SREBP to the cytosol.

Only the product of S1P cleavage can serve as a

substrate for S2P.

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Drugs used to inhibit cholesterol synthesis includecompetitive inhibitors of HMG-CoA Reductase.

Examples include various statin drugs such as lovastatin

(Mevacor) and derivatives (e.g., Zocor), Lipitor, etc.A portion of each statin is analogous in structure tomevalonate or to the postulated mevaldehydeintermediate.

Extensive clinical trials have shown that the statin drugsdecrease blood cholesterol and diminish risk ofcardiovascular disease.

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CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 S ProteinCCH2

CH3

farnesyl residue linked to protein via cysteine S

Since farnesyl & geranylgeranyl membrane anchorsare important for signal proteins that regulate cell cycleprogression, inhibitors of prenylating enzymes such asFarnesyl Transferase are being tested as anti-cancerdrugs.

However, toxic side effects may limit usefulness of thisapproach.