Cholesterol Synthesis. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the precursor for cholesterol synthesis. HMG-CoA is an intermediate on the pathway.

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.

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 then 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

Mevalonate formation:

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

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 (to be discussed later).

HMG-CoA Reductase has a cleavable membrane domain that links it to the ER. The isolated catalytic portion of the enzyme forms a tetramer, consisting of 2 dimers, each of which includes 2 active sites.

The binding site for HMG-CoA in one monomer is adjacent to the binding site for NADPH in the other. Explore this structure with Chime.

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 interconverts isopentenyl pyrophosphate & dimethylallyl pyrophosphate. The mechanism involves deprotonation and protonation.

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 elimination of PPi to yield a reactive carbocation.

Prenyl Transferase (Farnesyl Pyrophosphate Synthase) has been crystallized with the substrate geranyl pyrophosphate in the active site (Chime exercise).

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

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 oxidation of squalene to form 2,3-oxidosqualene. This mixed function oxidation requires NADPH as reductant & O2 as oxidant. One O atom is incorporated into

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

O

NADP+

O2 H2O

HO

H+NADPH

squalene 2,3-oxidosqualene lanosterol

Squalene Oxidocyclase catalyzes a series of cyclization reactions, initiated by donation of a proton to the epoxide.

The product is the sterol lanosterol.

O

NADP+

O2 H2O

HO

H+NADPH

squalene 2,3-oxidosqualene lanosterol

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

Additional modification of cholesterol yields various steroid hormones.

Many of these reactions are mixed function oxidations, requiring O2 & NADPH.

H O H O

lan o ste ro l ch o leste ro l

1 9 s tep s

In a mixed function oxidation, one O atom of O2 is

incorporated into a substrate & the other O atom reduced to H2O. E.g., hydroxylation catalyzed by cyt P450.

In a pathway associated with ER membranes, NADPH transfers 2e to cyt P450 via a Reductase, which has FAD &

FMN prosthetic groups. O2 binds to the reduced heme Fe of cyt P450, and hydroxylation is catalyzed.

2e

NADPH FAD/FMN P450

ROH + H2O

RH + O2

There are many variants of cytochrome P450. Some

have broad substrate specificity. Some are in mitochondria. Others are associated with ER membranes.

Substrates include steroids & non-polar xenobiotics (drugs & other foreign compounds). Detoxification involves reactions like hydroxylation that increase polarity, so compounds can be excreted by the kidneys.

X

N N

Fe

N N

Y

The heme prosthetic group of cyt P450 has a cysteine S as

axial ligand (X or Y). The other axial position, where O2 binds, may be open or have a bound H2O, that is displaced by O2 (Chime exercise).

Farnesyl pyrophosphate, an intermediate on the pathwayfor cholesterol synthesis, also serves as precursor forsynthesis of various isoprenoids: dolichol (role in synthesis of oligosaccharide chains of glycoproteins) coenzyme Q (ubiquinone, role in electron transfer chain) prenylated proteins (geranylgeranyl & farnesyl groups

anchor some proteins to membranes).

membrane

lipid anchor

protein

O

O

CH 3O

CH 3CH 3O

(CH 2 CH C CH 2)nH

CH 3

coenzym e Q

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.

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

The level of of HMG-CoA Reductase is modulated by regulated proteolysis. Degradation of HMG-CoA Reductase is stimulated

by oxidized derivatives of cholesterol, mevalonate, & farnesol (dephosphorylated farnesyl pyrophosphate).

The membrane domain of HMG-CoA Reductase has a sterol-sensing domain that may have a role in activation of the enzyme’s degradation.

Transcription factors called SREBPs (sterol regulatory element binding proteins), particularly SREBP-2, also respond to cell sterol levels.

SCAP (SREBP cleavage-activating protein) has a sterol-sensing domain similar to that of HMG-CoA Reductase. SCAP transports the SREBP precursor to the golgi, where protease S1P cleaves it. A 2nd protease S2P then cleaves in the membrane domain to release the N-terminal SREBP.

When sterol levels are low, SREBPs are released by cleavage of precursor proteins in ER membranes. SREBPs translocate into the nucleus where they activate transcription of genes for HMG-CoA Reductase & other cholesterol synthesis enzymes.

N C

membrane

cytosol

lumen

S2P cleavage releasing SREBP

SCAP-activated S1P cleavage

Pharmaceutical Intervention

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. In addition, it has been suggested that the ring structures of the statin drubs may associated with the NADPH binding site in the enzyme.