Download Cholesterol Synthesis

Molecular Biochemistry II
Cholesterol Synthesis
Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.
O

O
C
OH
CH2
C
O
CH2
C
SCoA
CH3
hydroxymethylglutaryl-CoA
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
H3C
H2O  O
H3C
C
CH2
C
SCoA
SCoA
HMG-CoA
Synthase
HSCoA
OH
O
O
C
acetoacetyl-CoA
acetyl-CoA

O
C
CH2
C
O
CH2
C
SCoA
CH3
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.
C
H2C
CH3
CH2
C

O
C
H2C
O
HMG-CoA
HMG-CoA
Reductase
2NADP+
+ HSCoA
HO

SCoA
O
O
2NADPH
C
CH3
CH2
C
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
CH3
C
Mevalonate is
phosphorylated by 2
sequential Pi transfers
from ATP, yielding
the pyrophosphate
derivative.
ATP-dependent
decarboxylation, with
dehydration, yields
isopentenyl
pyrophosphate.
H2C
CH2
mevalonate
C

O
O
HO

2 ATP
(2 steps)
2 ADP
CH3
C
H2C
CH2 OH
CH2 CH2
O
O
C
O
O
O
O
P
O
O
5-pyrophosphomevalonate
CO2
CH3
ATP
ADP + Pi
O
C
H2C
P
O
CH2 CH2
O
P
O
O
O
P
O
O
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.
H2
C
C
H2C
C
H2
O
O
P
O
O
O
isopentenyl pyrophosphate
CH3
C
H2C
C
H
isoprene
CH2
P
O
O
CH3
O
C
H2C
CH2 CH2
O
isopentenyl
pyrophosphate
P
O
O
P
O
O
O
O
O
CH3
C
H3C
CH
CH2
dimethylallyl
pyrophosphate
O
P
O
O
P
O
O
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
H3C
C
O
CH
CH2
O
O
P
O
P
O
O
O
CH3
dimethylallyl pyrophosphate
H2C
C
O
CH2
CH2
O
P
O
O
O
PPi
CH3
CH3
H3C
C
CH
CH2
CH2
C
CH2
O
P
O
O
P
O
CH3
H2C
O
O
O
geranyl pyrophosphate
C
O
CH2
CH2 O
P
O
O
O
CH3
H3C
C
CH
CH2
CH2
C
CH3
CH
CH2
P
O
O
isopentenyl pyrophosphate
PPi
CH3
O
isopentenyl pyrophosphate
O
CH
P
CH2 C
O
CH
CH2
O
P
O
O
P
O
farnesyl pyrophosphate
O
O
Each condensation involves a carbocation formed as PPi is eliminated.
CH3
CH3
2 H3C
C
CH
NADPH
CH2
CH2
C
CH3
CH
CH2
CH2 C
O
CH
CH2
2 farnesyl pyrophosphate
O
P
O
O
O
P
O
O
NADP+ + 2 PPi
NADP+
NADPH
O2
H2O
O
squalene
H+
2,3-oxidosqualene
HO
lanosterol
Squalene Synthase: Head-to-head condensation of 2 farnesyl
pyrophosphate, with reduction by NADPH, yields squalene.
NADP+
NADPH
O2
H2O
O
squalene
H+
2,3-oxidosqualene
HO
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+
O
2,3-oxidosqualene
HO
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.
RH + O2
2e
NADPH
FAD/FMN
P450
ROH + H2O
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.
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.
RH + O2

2e
NADPH
FAD/FMN
P450
ROH + 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
CH3
H3C
C
CH
CH2
CH2
C
CH3
CH
CH2
CH2 C
farnesyl pyrophosphate
O
CH
CH2
O
P
O
O
O
P
O
O
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
CH3
H3C
C
CH
CH2
CH2
C
CH3
CH
CH2
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
CH3
H3C
C
CH
CH2
CH2
C
CH3
CH
CH2
CH2 C
CH
CH2
S
Protein
farnesyl residue linked to protein via cysteine 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.
CH3
H
CH2
C
CH3
CH
CH2
CH2
CH
O
CH2
CH2
16-19
O
P
O
O
O
P
O
O
dolichol 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
(CH2 CH
O
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
CH
C
CH2
3
H
OH
O
N
HC
CH3
N

OOC
Fe
CH2 CH2
N
CH
N
CH2
CH2
CH3
CH2
COO
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.
ER lumen
The SREBP precursor
protein is embedded in the
endoplasmic reticulum
(ER) membrane via two
transmembrane a-helices.
membrane
SCAP
binding
domain C
cytosol
N
SREBP
domain
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
 Protease S1P (site one
protease), an integral
protein of golgi
membranes, cleaves the
SREBP precursor at a
site in the lumenal
domain.
SCAP-activated
S1P cleavage
membrane
C
N
S2P cleavage
releasing
SREBP
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.
PDB 1AM9
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
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.
CH3
CH3
H3C
C
CH
CH2
CH2
C
CH3
CH
CH2
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.