Molecular Biochemistry II

Cholesterol Synthesis

Contents of this page:
HMG-CoA formation and conversion to mevalonate

Conversion of mevalonate to isoprenoid precursors
Synthesis of squalene and its conversion to lanosterol
Conversion of lanosterol to cholesterol
Non-steroidal isoprenoids
Regulation of cholesterol synthesis and pharmaceutical intervention

Cholesterol synthesis:

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.

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

HMG-CoA Reductase catalyzes production of mevalonate from HMG-CoA. 

The carboxyl group of hydroxymethylglutarate that is in ester linkage to the thiol of coenzyme A is reduced first 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.

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 is rate-limiting for cholesterol synthesis. This enzyme is highly regulated and the target of pharmaceutical intervention (to be discussed later).

Explore at right the structure of the catalytic portion of HMG-CoA Reductase.
HMG-CoA Reductase
Mevalonate is phosphorylated by 2 sequential phosphate transfers from ATP, yielding the pyrophosphate derivative. 

Pyrophosphomevolanate Decarboxylase catalyzes ATP-dependent decarboxylation, with dehydration, to yield 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.

Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate and dimethylallyl pyrophosphate

The mechanism involves protonation followed by deprotonation.

Prenyl Transferase catalyzes a series of head-to-tail condensation reactions.

Dimethylallyl pyrophosphate reacts with isopentenyl pyrophosphate 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.

Prenyl Transferase (Farnesyl Pyrophosphate Synthase) has been crystallized with the substrate geranyl pyrophosphate bound at the active site. Explore its structure at right.


Prenyl Transferase

Squalene Synthase catalyzes head-to-head condensation of 2 molecules of farnesyl pyrophosphate, with reduction by NADPH, to yield squalene

Squalene epoxidase catalyzes oxidation of squalene to form 2,3-oxidosqualene. This mixed function oxidation requires NADPH as reductant and O2 as oxidant. One atom of oxygen is incorporated into the substrate (as the epoxide) and the other oxygen atom is reduced to water.

Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization (diagram p. 950). 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 of the cyclization reaction is the sterol lanosterol.

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes associated with endoplasmic reticulum membranes (p. 952).
Additional modifications yield the various steroid hormones or vitamin D (p. 958). 

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 cytochrome 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. 

Cytochrome P450 enzymes catalyze various oxidative reactions. Many are mixed function oxidations (mono-oxygenations), that require O2 as well as a reductant such as NADPH. One oxygen atom is incorporated into a substrate and the other oxygen atom is reduced to water.

An example is hydroxylation of a steroid, as in the endoplasmic reticulum electron transfer pathway depicted at right, NADPH transfers 2 electrons to cytochrome P450 via a reductase that has FAD and FMN prosthetic groups.

A cysteine S atom typically serves as an axial ligand (X or Y at right) for the iron atom of a cytochrome 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 heme iron of cyt P450. In the example above, one oxygen atom is reduced to water, and a substrate is hydroxylated.

For a diagram of the reaction cycle, see article by M. Coon (requires Annual Reviews subscription).

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

P450 substrates include steroids, polyunsaturated fatty acids, eicosanoids, retinoids, and various non-polar xenobiotics (drugs and 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 at right the structure of the hemoprotein domain of a Bacillus magaterium cytochrome P450.


Cytochrome P450

 
Isoprenoids: Farnesyl pyrophosphate, an intermediate on the pathway for cholesterol synthesis, 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 those proteins to membranes (p. 403). Many proteins involved in cell signaling have such lipid anchors, including small GTP-binding proteins such as Ras.

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 three 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 (p. 853-854). Additional roles have been proposed; dolichol is found in many membranes of cells.

Coenzyme Q (ubiquinone), which has an isoprenoid side-chain, functions in the electron transfer chain (p. 810, 823).

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

Regulation of cholesterol synthesis 

HMG-CoA Reductase, the rate-determining 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 

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

The SREBP precursor protein is embedded in the endoplasmic reticulum (ER) membrane via two transmembrane a-helices (diagram at right). 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).
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. Association with Insig causes the SREBP-SCAP precursor complex to be retained within the ER.

When sterol levels are low, SCAP and Insig do not interact. This allows the SCAP-SREBP precursor complex to translocate from the ER to the golgi apparatus.

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

An intramembrane zinc metalloprotease domain of another golgi protease S2P (site 2 protease) 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 and degraded (see notes on ubiquitin & the proteasome).

 

For additional diagrams see:

p. 955 Voet & Voet textbook;

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


Homodimeric DNA-binding domain of SREBP interacting with a sterol regulatory element DNA segment. Data of A. Parraga, L. Bellsolell, A. R. Ferre-D'Amare & S. K. Burley, 1998.

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 (p. 957). Extensive clinical trials have shown that the statin drugs decrease blood cholesterol and diminish risk of cardiovascular disease.

Since  farnesyl and geranylgeranyl membrane anchors are important for signal proteins that regulate progression through the cell cycle, 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.

Copyright 1998-2008 by Joyce J. Diwan. All rights reserved.

Additional material on Cholesterol Synthesis:
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