Molecular Biochemistry II

Cholesterol Synthesis

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

O


OH CH2 C CH3 CH2

O C SCoA

hydroxymethylglutaryl- o

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.

O
H 2O 

O CH2 C SCoA

O C

H3C SCoA HSCoA O

acetoacetyl-CoA HMG-CoA Synthase

OH CH2 C CH3 CH2 O C SCoA

acetyl-CoA

H3C

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.

HO

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.

CH3 C H2C C


CH2 O

C O

SCoA

HMG-CoA HMG-CoA Reductase

CH3

2NADPH 2NADP+ + HSCoA HO C H2C C



CH2 O

H2 C OH

mevalonate

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 ratelimiting for cholesterol synthesis. This enzyme is highly regulated and the target of pharmaceutical intervention.

HO C

CH3 CH2 CH2 OH

Mevalonate is phosphorylated by 2 sequential Pi transfers from ATP, yielding the pyrophosphate derivative. ATP-dependent decarboxylation, with dehydration, yields isopentenyl pyrophosphate.


H2C C


mevalonate
O 2 ATP (2 steps) 2 ADP O O P O ATP ADP O CH2 CH2 O P O O O O P O O

HO C H2C C O O

CH3 CH2 CH2

5-pyrophosphomevalonate
CO2 Pi O P O O

CH3 C H2C

isopentenyl pyrophosphate

CH3

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

C H2 C C H2

H2 C O

O P O O

O P O O

isopentenyl pyrophosphate

CH3 C H2 C C H CH2

isoprene

CH3 C H2 C CH2 CH2 O O P O O O P O O

isopentenyl pyrophosphate

CH3 C H3 C CH CH2 O O P O O O P O O

dimethylallyl pyrophosphate

Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate & dimethylallyl pyrophosphate. Mechanism: protonation followed by deprotonation.

Condensation Reactions

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.

CH3 H3 C C CH CH2 O

O P O O

O P O H2 C PPi O CH3 C CH2 CH2 O O P O O O P O O

dimethylallyl pyrophosphate

isopentenyl pyrophosphate
O O O P O O CH2 CH2 O P O O O P O O O P O CH3

CH3 H3 C C CH CH2 CH2

CH3 C CH CH 2 O

geranyl pyrophosphate
H2 C PPi CH3 H3 C C CH CH2 CH2 CH3 C CH CH2

isopentenyl pyrophosphate
CH3 O CH CH2 O P O O P O

CH2 C

arnesyl pyrophosphate O O Each condensation involves a carbocation ormed as PPi is eliminated.

CH3

CH3 CH CH2 CH2 C CH CH2

CH3 CH2 C CH CH2 O

O P O O

O P O O

2 H3C

NADPH NADP+ + 2 PPi

2 farnesyl pyrophosphate

NADP+ NADPH

H+

O2

H2O

HO

squalene

2,3-oxidosqualene

lanosterol

Squalene Synthase: Head-to-head condensation of 2 farnesyl pyrophosphate, with reduction by NADPH, yields squalene.

NADP+ NADPH

H+

O2

H2O

HO

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.

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

H+

HO

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.

19 steps
HO HO

lanosterol

cholesterol

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes in ER membranes. Additional modifications yield the various steroid hormones or vitamin D.

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.

2e NADPH

RH + O2 FAD/FMN P450 R + H2 O OH

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.

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.

2e NADPH FAD/FMN P450

RH + O2

OH R + H2O

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.

 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.

CH3 H3C C CH CH2 CH2

CH3 C CH CH2

CH3 CH2 C CH CH2 O

O P O O

O P O O

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.

CH3 H3C C CH CH2 CH2

CH3 C CH CH2

CH3 CH2 C CH CH2 S

Protein

farnesyl residue linked to protein via cysteine S

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.

protein lipid anchor

membrane

CH3 H3C C CH CH2 CH2

CH3 C CH CH2

CH3 CH2 C CH CH2 S

Prot i

arnesyl residue linked to protein via cysteine

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.

CH3 H CH2 C CH CH2 CH2

16-19

CH3 CH CH2 CH2 O

O P O O

O P O O

olichol pyrophosphate

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.

O CH3O CH3 CH3 CH3O O (CH2 CH C CH2)nH

coenzyme Q

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

CH3 CH2 CH3 HC CH2 OH CH C CH2

3

O N HC N


CH3 Fe N N CH CH2

OOC

CH2 CH2

CH2 CH2 COO

CH3

Heme a

 Heme a, a constituent of respiratory chain complexes, has a farnesyl side-chain.

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.

E lumen

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

membrane E domain

binding domain C cytosol

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 cleavageactivating protein).

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 (in ER) sterol PreSREBP-SCAP-Insig Insig PreSREBP-SCAP (translocates to golgi)

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.

golgi lumen

S P-activated S1P cleavage

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

membrane S2P cleavage releasing SREBP

N cytosol

 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 D comple

PDB

Homodimeric D binding domain of SREBP interacting with a sterol regulatory element segment. D

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.

CH3 H3C C CH CH2 CH2

CH3 C CH CH2

CH3 CH2 C CH CH2 S

Protein

farnesyl residue linked to protein via cysteine 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.