cholesterol synthesis. hydroxymethylglutaryl-coenzyme a (hmg-coa) is the precursor for cholesterol...
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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.