Cholesterol Synthesis

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• Cholesterol Synthesis

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• 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.

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• 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.

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• 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.

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• 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.

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• Mevalonate is phosphorylated by 2 sequential Pi
  transfers from ATP, yielding the pyrophosphate
  derivative.
• ATP-dependent decarboxylation, with dehydration,
  yields isopentenyl pyrophosphate.

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• Isopentenyl pyrophosphate is the first of several
  compounds in the pathway that are referred to as
  isoprenoids, by reference to the compound
  isoprene.

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• Isopentenyl Pyrophosphate Isomerase interconverts
  isopentenyl pyrophosphate dimethylallyl
  pyrophosphate. The mechanism involves
  deprotonation and protonation.

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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
  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).

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• Squalene Synthase Head-to-head condensation of 2
  farnesyl pyrophosphate, with reduction by NADPH,
  yields squalene.

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• 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.

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• Squalene Oxidocyclase catalyzes a series of
  cyclization reactions, initiated by donation of a
  proton to the epoxide.
• The product is the sterol lanosterol.

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• 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.

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• 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.

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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).

• 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.

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• Farnesyl pyrophosphate, an intermediate on the
  pathway
• for cholesterol synthesis, also serves as
  precursor for
• synthesis 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).

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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.

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

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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.

• 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.

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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.