Molecular Biochemistry II

Cholesterol Synthesis

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

HMG-CoA is 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. 

A carboxyl group of hydroxymethylglutarate is reduced to an alcohol, with NADPH serving as reductant in a 2-step reaction. Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA.

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. 

ATP-dependent decarboxylation, with dehydration, yields 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 interconverts isopentenyl pyrophosphate and dimethylallyl pyrophosphate

The mechanism involves protonation and deprotonation.

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

Dimethylallyl pyrophosphate reacts with isopentenyl pyrophosphate to form geranyl pyrophosphate

The subsequent reaction yields farnesyl pyrophosphate.

Each condensation reaction is thought to involve elimination of PPi to yield a reactive carbocation. 

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

Head-to-head condensation of 2 molecules of farnesyl pyrophosphate, with reduction by NADPH, yields squalene

Squalene epoxidase catalyzes oxidation of squalene to form 2,3-oxidosqualene. The 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 reduced to water.

Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by donation of a proton to the epoxide, that lead to cyclization (p. 603). The product is the sterol lanosterol .

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes associated with endoplasmic reticulum membranes. Additional modification of cholesterol yields various steroid hormones (p. 229). Many of these reactions are mixed function oxidations, requiring O2 and NADPH

In a mixed function oxidation, one oxygen atom of O2 is incorporated into a substrate and the other oxygen atom is reduced to water. An example is hydroxylation of a substrate, catalyzed by cytochrome P450:

In a pathway associated with endoplasmic reticulum membranes, NADPH transfers 2 electrons to cyt P450 via a Reductase, which has FAD and FMN prosthetic groups. O2 binds to the reduced heme iron of cyt P450, and hydroxylation is catalyzed.

The heme prosthetic group of cyt P450 has a cysteine S as an axial ligand (X or Y in the diagram at right). The other axial position, where O2 binds, may be open or have a bound H2O, that is displaced by O2.

There are many variants of cytochrome P450. Some have broad substrate specificity. Some are localized in mitochondria. Others are associated with endoplasmic reticulum membranes. Substrates include steroids and various non-polar xenobiotics (drugs and other foreign compounds). Detoxification involves reactions such as hydroxylation that increase polarity, so that compounds can be excreted by the kidneys.

View the structure of the hemoprotein domain of Bacillus magaterium cytochrome P450, a protein analogous to cytochrome P450 of endoplasmic reticulum:

Cytochrome P450

Farnesyl pyrophosphate, an intermediate on the pathway for cholesterol synthesis, also serves a precursor for synthesis of various isoprenoids:

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 is by varied transcription and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

SCAP (SREBP cleavage-activating protein), which also has a sterol-sensing domain, activates protease S1P (site one protease) that cleaves in  the lumenal domain of the SREBP precursor protein. Another protease, S2P, then cleaves the transmembrane domain to release SREBP, the N-terminal domain of the precursor protein.

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).  A portion of each of these compounds is analogous in structure to mevalonate (p. 607). It has been suggested that the ring structures of the statin drugs may associate with the NADPH binding site in the enzyme.

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Copyright 1998-2001  by Joyce J. Diwan.   All rights reserved.

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