Cholesterol Synthesis

Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved.

Molecular Biochemistry II

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

HMG-CoA is also an intermediate on the pathway for synthesis of ketone bodies from acetyl-CoA.

The enzymes for ketone body production are located in the mitochondrial matrix.

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

CH2 C CH2 C

OH O

SCoA

CH3

C

O

O

hydroxymethylglutaryl-CoA

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

The carboxyl of HMG that is in ester linkage to the CoA thiol is reduced to an aldehyde, and then to an alcohol.

NADPH serves as reductant in the 2-step reaction.

Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA.

+ HSCoA

H2CC

CH3HO

CH2

CO O

C SCoA

O

H2CC

CH3HO

CH2

CO O

H2C OH

2NADP+

2NADPH

HMG-CoA

mevalonate

HMG-CoAReductase

HMG-CoA Reductase is an integral protein of endoplasmic reticulum membranes.

The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme.

The HMG-CoA Reductase reaction, in which mevalonate is formed from HMG-CoA, is rate-limiting for cholesterol synthesis.

This enzyme is highly regulated and the target of pharmaceutical intervention.

Mevalonate is phosphorylated by 2 sequential Pi transfers

from ATP, yielding the pyrophosphate derivative.

ATP-dependent decarboxylation, with dehydration, yields isopentenyl pyrophosphate.

H2CC

CH3HO

CH2

C O O

CH2 OH

H2C

C

CH2 CH2 O P O P O

O

O

O

O

CH3

H2CC

CH3HO

CH2

C O O

CH2 O P O P O

O

O

O

O

CO2

ATP

ADP + Pi

2 ATP

2 ADP

mevalonate

5-pyrophosphomevalonate

(2 steps)

isopentenyl pyrophosphate

Isopentenyl pyrophosphate is the first of several compounds in the pathway that are referred to as isoprenoids, by reference to the compound isoprene.

isoprene

H2CC

CCH2

CH3

H

is o p e n te n y l p y ro p h o s p h a te

H 2 CC

CH 2

H 2C

C H 3

O P

O

O

O P O

O

O

Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate & dimethylallyl pyrophosphate.

Mechanism: protonation followed by deprotonation.

H2C

C

CH2 CH2 O P O P O

O

O

O

O

CH3

H3C

C

CH CH2 O P O P O

O

O

O

O

CH3

isopentenyl pyrophosphate

dimethylallyl pyrophosphate

Prenyl Transferase catalyzes head-to-tail condensations:

Dimethylallyl pyrophosphate & isopentenyl pyrophosphate 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

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

farnesyl pyrophosphate

Each condensation involves a carbocation formed as PPi is eliminated.

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+ + 2 PP i

NADPH

2 farnesyl pyrophosphate

squalene 2,3-oxidosqualene lanosterol

Squaline epoxidase catalyzes conversion of squalene to 2,3-oxidosqualene.

This mixed function oxidation requires NADPH as reductant & O2 as oxidant. One O atom is incorporated into substrate (as the epoxide) & the other O is reduced to water.

O

NADP+

O2 H2O

HO

H+NADPH

squalene 2,3-oxidosqualene lanosterol

Structural studies of a related bacterial enzyme have confirmed that the substrate binds at the active site in a conformation that permits cyclization with only modest changes in position as the reaction proceeds.

The product is the sterol lanosterol.

O HO

H+

2,3-oxidosqualene lanosterol

Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization.

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes in ER membranes.

Additional modifications yield the various steroid hormones or vitamin D.

H O H O

lan o ste ro l ch o leste ro l

1 9 s tep s

Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P450 enzyme superfamily.

The human genome encodes 57 members of the cyt P450 superfamily, with tissue-specific expression and intracellular localization highly regulated.

Some P450 enzymes are localized in mitochondria.

Others are associated with endoplasmic reticulum membranes.

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 & the other oxygen atom is reduced to water.

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

NADPH transfers 2 electrons to cytochrome P450 via a reductase that has FAD & FMN prosthetic groups.

2e

NADPH FAD/FMN P450

ROH + H2O

RH + O2

X

N N

Fe

N N

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 O2 binds, may be open or have a bound H2O that is displaced by O2.

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

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

P450 substrates include steroids, polyunsaturated fatty acids, eicosanoids, retinoids, & various non-polar xenobiotics (drugs & other foreign compounds). Some P450 enzymes have broad substrate specificity.

Mechanisms for detoxification of non-polar compounds include reactions such as hydroxylations that increase polarity, so that the products of these reactions can be excreted by the kidneys.

Explore with Chime the hemoprotein domain of a Bacillus magaterium cytochrome P450.

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 pathway for cholesterol synthesis, also serves also as precursor for synthesis of various non-steroidal isoprenoids.

The importance of the other products of the pathway that originates with mevalonate is reflected in serious diseases that result from genetic defects in this pathway.

Prenylated proteins have covalently linked geranylgeranyl or farnesyl groups that anchor them to membranes.

Many proteins involved in cell signaling have such lipid anchors, including small GTP-binding proteins such as Ras.

membrane

lipid anchor

protein

C H C H 2CH 3 C

C H 3

C H C H 2CC H 2

C H 3

C H C H 2 S P r o t e i nCC H 2

C H 3

f a r n e s y l r e s i d u e l i n k e d t o p r o t e i n v i a c y s t e i n e S

C H C H 2CH 3 C

C H 3

C H C H 2CC H 2

C H 3

C H C H 2 S P r o t e i nCC H 2

C H 3

f a r n e s y l r e s i d u e l i n k e d t o p r o t e i n v i a c y s t e i n e S

Farnesyl Transferase catalyzes transfer of the farnesyl moiety of farnesyl pyrophosphate to a cysteine residue in a sequence CaaX at the C-terminus of a protein, "a" being an aliphatic amino acid.

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

Some other isoprenoids:

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

Additional roles have been proposed; dolichol is found in 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

dolichol pyrophosphate

Coenzyme Q (ubiquinone), which has an isoprenoid side-chain, functions in the electron transfer chain.

O

O

CH 3O

CH 3CH 3O

(CH 2 CH C CH 2)nH

CH 3

coenzym e Q

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

Regulation of cholesterol synthesis

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

Short-term regulation:

HMG-CoA Reductase is inhibited by phosphorylation, catalyzed by AMP-Dependent Protein Kinase (which also 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 expended in synthesizing cholesterol.

Long-term regulation is by varied formation and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:

• Degradation of HMG-CoA Reductase is stimulated 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).

Regulated transcription:

• A family of transcription factors designated SREBP (sterol regulatory element binding proteins) regulate synthesis of cholesterol and fatty acids.

Of these, SREBP-2 mainly regulates cholesterol synthesis. (SREBP-1c mainly regulates fatty acid synthesis.)

• When sterol levels are low, SREBP-2 is released by cleavage of a membrane-bound precursor protein.

• SREBP-2 activates transcription of genes for HMG-CoA Reductase and other enzymes of the pathway for cholesterol synthesis.

The N-terminal SREBP domain, which extends into the cytosol, has transcription factor capability.

The C-terminal domain, also on the cytosolic side of the membrane, interacts with a cytosolic domain of another ER membrane protein SCAP (SREBP cleavage-activating protein).

The SREBP precursor protein is embedded in the endoplasmic reticulum (ER) membrane via two transmembrane -helices.

N C

membrane

cytosol

ER lumen

SREBP domain

SCAP binding domain

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 to translocate from the ER to the golgi apparatus.

SCAP has a transmembrane sterol-sensing domain homologous to that of HMG-CoA Reductase.

When bound to a sterol, the sterol-sensing domain of SCAP binds the ER membrane protein Insig.

PreSREBP-SCAP/sterol-Insig

sterol

PreSREBP-SCAP-Insig

Insig

PreSREBP-SCAP (translocates to golgi)

(in ER)

Protease S1P (site one protease), an integral protein of golgi membranes, cleaves the SREBP precursor at a site in the lumenal domain.

N C

membrane

cytosol

golgi lumen

S2P cleavage releasing SREBP

SCAP-activated S1P cleavage

An intramembrane zinc metalloprotease domain of another golgi protease S2P then catalyzes cleavage within the transmembrane segment of the SREBP precursor, releasing SREBP to the cytosol.

Only the product of S1P cleavage can serve as a substrate for S2P.

The released SREBP enters the cell nucleus where it functions as a transcription factor to activate genes for enzymes of the cholesterol synthesis pathway.

Its lifetime in the nucleus is brief, because SREBP is ubiquitinated & degraded.

Diagram (in article by P. J. Espenshade; requires J. Cell Sci. subscription)

SREBP-DNA complex

PDB 1AM9

Homodimeric DNA-binding domain of SREBP interacting

with a sterol regulatory element DNA segment.

Drugs used to inhibit cholesterol synthesis include competitive 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 to mevalonate or to the postulated mevaldehyde intermediate.

Extensive clinical trials have shown that the statin drugs decrease blood cholesterol and diminish risk of cardiovascular disease.

C H C H 2CH 3 C

C H 3

C H C H 2CC H 2

C H 3

C H C H 2 S P r o t e i nCC H 2

C H 3

f a r n e s y l r e s i d u e l i n k e d t o p r o t e i n v i a c y s t e i n e S

Since farnesyl & geranylgeranyl membrane anchors are important for signal proteins that regulate cell cycle progression, inhibitors of prenylating enzymes such as Farnesyl Transferase are being tested as anti-cancer drugs.

However, toxic side effects may limit usefulness of this approach.