BCH3001L Biochimie Métabolique 2

BCH3001L Biochimie Métabolique 2

Prof Adriana E. MIELE Institut des Sciences Analytiques adriana.miele@univ-lyon1.fr

Planning of lectures

• The crossroads of intermediate Metabolism
• Photosynthesis
• Pentoses phosphate pathway (PPP)
• Nucleic acids metabolism
• Vitamins, cofactors & coenzymes
• Cholesterol & steroid hormones metabolism
• A case study: the nervous tissue

Cholesterol metabolism

• Cholesterol, bile salts and steroid hormones are a class of lipids present almost exclusively in eukaryotes
• The common part is the polycyclic non-aromatic ring: cyclopentane-perihydro-phenanthrene
• Cholesterol is the precursor of all this class of lipids in mammals

Cholesterol

• Cholesterol is a C27 cyclic hydrocarbon
• The 4 non aromatic rings make the molecule rigid
• When cholesterol is inserted into the membrane bilayer, the fluidity changes
• The alcoholic -OH group on C3 is the only

hydrophilic part of the molecule

Cholesterol synthesis

• The synthesis of cholesterol is carried out in liver only (cytosol and endoplasmic reticulum, ER)
• It can be divided into 4 steps, each subdivided by several reactions:

• Condensation of 3 acetyl-CoA (C2) into mevalonate (C6)
• Oxidative decarboxylation and phosphorylation of mevalonate into isopentenyl pyrophosphate  (IPP, C5)
• Polymerization of 6 IPP into squalene (C30)
• Cyclisation of squalene into lanosterol; elimination of 3C to form cholesterol (C27)

Cholesterol synthesis:

2 Ac-CoA → acetoacetyl-CoA + CoA[pic 1]

Acetoacetyl-CoA + Ac-CoA → 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) + CoA

HMG-CoA + 2 NADPH,H+ → mevalonate + CoA +2 NADP+

• The first 2 reactions are in common with the ketonic corps production → same enzymes → thiolase + HMG-CoA synthase
• The 3rd is catalyse by HMG-CoA reductase → key checkpoint and pharmacologic target (statines)

Cholesterol synthesis:

In the cytosol

synthesis:

On the ER membrane

(endoplasmic reticulum)

Mevalonate 

Voet & Voet, Biochemistry

Cholesterol synthesis: 2nd step

Mevalonate + 2 ATP → 5-PP-mevalonate + 2 ADP[pic 2]

5-PP-mevalonate + ATP → Isopentenyl-PP + ADP + Pi + CO2

• The second step consumes 3 ATP to synthesise an intermediate of high energy content and 5C → IPP
• IPP is both an intermediate of cholesterol and a donor of isoprenoid groups for post-translational modifications → geranylation et farnesylation of proteins to be inserted into membrane bilayers

synthesis: 2nd step

Stryer, Biochemistry

• 3-phosphoryl-5-PP-mevalonate is an intermediate of decarboxylation
• Marking the -OH in position 3 proved its elimination with the phosphate group
• CO2 is instantaneously transformed into bicarbonate by cytosolic carbonic anhydrase

synthesis: 3rd

2 IPP → geranyl-PP + 2 Pi

Geranyl-PP + IPP → farnesyl-PP + 2 Pi

2 F-PP + NADPH,H+ → squalene + 2 Pi + NADP+

• The consecutive polymerisation of IPP units is catalysed by prenyltransferase
• Each IPP contains a double bond which makes the

30C skeleton rigid and limits the number of possible conformations

synthesis: 3rd step

• Actually, prenyltransferase does not condesate 2

IPP, byt 1 IPP and 1 DAPP (dimethyl-allyl-PP)

• DAPP is formed by IPP-isomérase that uses the catalytic couple Glu-Cys as H+ acceptor/donor

synthesis: 3rd

Cholesterol synthesis: 4th step

Squalene + O2 + NADPH,H+ → lanosterol + H2O + NADP+

Lanosterol + 19 O2 + 19 NADPH,H+ → cholesterol + 19 H2O +

19 NADP+ + 2 CO2 + HCOOH (in 19 reactions)

• Squalene is converted into cyclic lanosterol by squalene epoxydase and oxosqualene cyclase
• Lanosterol is the common C30 molecules which gives rise to cholesterol, biliary salts and steroid hormones

synthesis: 4th

[pic 3]

        19 NADP+        Stryer, Biochemistry

2 CO2

HCOOH

Cholesterol synthesis: a summary

[pic 4]

Energetic balance of cholesterol biosynthesis

● To synthesise 1 molecule of cholesterol liver cells need:

• 18 AcCoA (3 AcCoA for IPP and 6 IPP in total) → β-oxidation or glycolysis
• 32 NADPH,H+ (2 for each IPP + 20 for cyclisation) → PPP
• Min 18 ATP (3 for each IPP) → CRM

[pic 5]

Regulation of cholesterol synthesis

• The metabolic checkpoint is mevalonate synthesis by HMG-CoA réductase
• This is an integral membrane protein present on the rough ER membrane
• HMG-R is subjected to a regulation:

• Short term: inhibition by mevalonate & cholesterol; posttranslational modification
• long term: gene transcription and protein degradation

• Objective: keep the blood circulating [cholestérol] constant ≈ 800 mg/jour

HMG-R short term regulation

• Mevalonate is a competitive inhibitor; cholesterol is an allosteric inhibitor →  direct inhibition by product and final metabolite
• HMG-R can be phosphorylated → inactive:

• Insulin and Thyroid hormone block phosphorylation → reductase active
• Glucagon and glucocorticoids activate phosphorylation → reductase inactive

• A low energetic charge (⭣[ATP]) inhibits HMG-R

HMG-CoA reductase regulation

[pic 6]

Long term regulation of HMG-R

• Protein quantity is regulated at transcriptional and post-translational level by steroid hormones:

• HMG-R has the catalytic part into the cytosol and the regulatory domain into the RER membrane → steroid hormones can bind in the membrane, change the conformation and induce proteolysis
• Steroid hormones can also bind to a sensor protein

(SREBP), anchored on the RER. Without hormones, SREBP is transferred into the nucleus → binds the DNA sequence SRE and activates HMG-R transcription

Long term HMG-R regulation

Voet & Voet, Biochemistry[pic 7]

• SREBP : sterol regulatory element binding protein
• SCAP: SREBP cleavage activating protein → steroid hormones sensor
• Insig: regulator of translocation between RER and Golgi

Long term regulation of HMG-R

• In the absence of sterols, Insig binds [pic 8]

COPII →  SCAP-

SREBP complex is translocated to Golgi

• In Golgi, 2 proteases, S1P + S2P, digest

SREBP by freeing the

DNA binding domain

(bHLH)

• bHLH translocates into the nucleus, binds to

SRE and activates

Voet & Voet, Biochemistry        genes transcription

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