Biosynthesis of Sterols

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2 sources of cholesterol:: 1. Diet
2. De novo synthesis in all cells
4 predominant tissues of Sterol biosynthesis:: 1. Liver
2. Adrenal cortex
3. Intestine
4. Reproductive tissues
Main sites where cholesterol functions: (2): 1. Cell membrane component
2. CNS/brain myelinated structures
2 Direct Derivatives of Cholesterol: 1. Steroid hormones
2. Bile acids
8 Derivatives of Cholesterol INTERMEDIATES:: -Vitamens D/E/A/K
-Carotenoids
-Rubber
-Plant hormones
-Phytol chain of chlorophyll
-Dolichols
-Ubiquinone/Plastiquinone
-Isoprene
Plant hormones (2):: -Abscisic Acid
-Gibberellic acid
3 Most important derivatives of cholesterol to remember:: -Steroid hormones
-Bile acids
-Vitamin D
In what form is cholesterol in membranes?: Unesterified - FREE
What distinguishes free unesterified cholesterol from esterified?: An -OH instead of Acyl
What enzyme makes free cholesterol esterified?: ACAT - Acyl-CoA Cholesterol Acyl Transferase
What reaction does ACAT catalyze?: Incorporates Fatty AcylCoA into Cholesterol w/ subsequent release of CoASH.
When does ACAT work? Why?: When membranes have too much free cholesterol ACAT esterifies it for intracellular storage or lipoprotein transfer.
What is the function of cholesterol in membranes?: Regulates fluidity and lateral mobility of proteins in the lipid bilayer.
What is the storage form of cholesterol called?: Cholesteryl Ester
What type of molecule is cholesterol?: Amphipathic
Nonpolar = 4 hydrocarbon rings
Polar head = OH
How many carbons are in cholesterol?: 27
What allows cholesterol to circulate in the blood?: Apolipoproteins
What is the carbon source for cholesterol synthesis de novo?: Acetyl CoA
3 Sources of AcCoA:: 1. FA beta-oxidation (mitochon)
2. Ketogenic amino acid oxidation (leucine/isoleucine)
3. PDH reaction
PDH Cofactors:: -TPP
-Lipoamide
-FAD
Reaction of PDH:: Pyruvate -> AcCoA
What enzyme catalyzes the COMMITTED STEP of Cholesterol biosynthesis?: HMG-CoA Reductase
What are the 4 major stages of cholesterol biosynthesis?: 0. Acetate
1. Mevalonate
2. Activated Isoprenes
3. Squalene
4. Cholesterol
What happens in Cholesterol biosynthesis Stage 1? Enzymes?: AcCoA -> Mevalonate
-3 reactions
-Thiolase, HMG-CoA Synthase, HMG-CoA Reductase
What is important re: first 2 reactions in Mevalonate synth?: They are shared with Ketogenesis
What is different about Ketogenesis vs. Mevalonate?: Ketogenesis = mitochondria
Cholest Synth = Cytosol

Hence dif. pools of enzymes
What are the enzymes shared by Ketogenesis & Mevalonate synth?: -Thiolase
-HMG-CoA Synthase
In what tissue are the cytosolic and mitosolic pools found?: Liver Parenchymal cells
Why is Mevalonate synthesis so very important?: Its 3rd reaction is the Commitment step of cholesterol biosynthesis
Where do HMG-CoA reductase and subsequent reactions occur?: Probably in peroxisomes.
What occurs in the 1st reaction of Mevalonate synthesis? Enzyme?: 2 AcCoA condensation - releases one CoASH
-Via Thiolase
What is the product of Thiolase?: Acetoacetyl-CoA
What occurs in the 2nd reaction of Mevalonate synth? Enzyme?: Acetoacetyl-CoA is condensed with another AcCoA
-Via HMG-CoA synthase
What is the product of HMG-CoA Synthase?: HMG - b-hydroxy-b-methylgutaryl-CoA
What occurs in the 3rd reaction of Mevalonate synth? Enzyme?: CoASH released from far end of HMG-CoA; C=O reduced to CH2-OH; the protons donated by 2NADPH;
-Via HMG-CoA Reductase
Where is HMG-CoA reductase found, and how is it situated?: -Integral - in the cell membrane
-Active site on cytosolic side
Product of HMG-CoA reductase is:: Mevalonate
What does Mevalonate get converted to? How?: 5-Carbon Activated isoprenes by Decarboxylation
How many reactions and enzymes are needed for stage 2 of cholesterol biosynthesis?: 4 Reactions/3 enzymes
Enzymes in 5C Activated Isoprene synthesis:: 1. Mevalonate 5-phosphotransferase
2. Phosphomevalonate kinase
3. Pyrophosphomevalonate Decarboxylase
Key thing to remember about stage 2 of cholest. biosynth:: IT REQUIRES 3 total ATP
What happens in the 1st reaction of stage 2?: PO4 added to terminal carbon of Mevalonate - replaces the -OH created by HMGCoA reductase.
What happens in the 2nd reaction of stage 2?: Add another PO4 right onto the one added in reaction 1.
What happens in the 3rd reaction of stage 2?: Add another PO4 onto the beta carbon of pyrophosphomevalonate.
What enzymes catalyze reactions 1 and 2?: 1. Mevalnt 5-phosphotransferase
2. Phosphomevalonate kinase
What enzyme catalyzes reactions 3 and 4 of stage 2?: Pyrophosphomevalonate decarboxylase - same enzyme for both reactions.
What occurs in reaction 4 of stage 2?: Decarboxylation of carbon 1 and loss of PO4 from carbon 3
What results from decarboxylating 3-Phospho-5-pyrophosphomevalonate?: 2 isomers:
-d3-isopentenyl pyrophosphate -Dimethylallyl pyrophosphate
What happens in Stage 3 of cholesterol biosynthesis? (in broad terms): Condensation of 6 activated 5-C isoprene units to make Squalene
What substrate is used in stage 3?: 1 NADPH
So substrates for: -Stage 1 -Stage 2 -Stage 3: 1 = 2 NADPH
2 = 3 ATP
3 = 1 NADPH
How many steps are entailed in Stage 3 of cholesterol synth?: 3 steps: C5 -> C10 -> C15 -> C30
What enzymes are used in Stage 3?: -Prenyl transferase (Rxns 1/2)
-Squalene synthase (Rxn 3)
What terms describe the nature of the 3 condensation reactions?: Rxns 1/2 = head-to-tail

Rxn 3 = head-to-head
What are the intermediates in Squalene synthesis? How many Cs?: 1. Geranyl PPi (10 C)
2. Farnesyl PPi (15 C)
What is important about Farnesyl PPi?: Used in post-translational protein modification
Where does Farnesylation occur on proteins?: C-terminal Cysteine residue
What is unique about the final condensation of 2 Farnesyl PPi?: -Head-to-Head
-Requires NADPH
What enzyme catalyzes the final step of stage 3?: Squalene synthase
How many carbons are in squalene? How many in cholesterol?: Squalene = 30

Cholesterol = 27
What needs to happen to squalene to make cholesterol?: Cyclization to close rings
In Squalene-Cholest conversion: -How many reactions? -How many enzymes? -What substrates?: -13 reactions
-11 enzymes
-1 NADPH
How does Squalene cyclization get started?: By activating it to Squalene Epoxide
What enzyme makes Squalene 2,3-Epoxide?: Squalene monooxygenase
What does Squalene monooxygenase require?: 1 molecule of O2
1 NADPH
What enzyme cyclizes Squalene 2,3-Epoxide?: Oxidosqualene cyclase
What is the product of oxidosqualene cyclase action?: Lanosterol
What needs to happen to Lanosterol to make cholesterol? (3 things): -Demethylation of 3 Carbons
-Reduce a double bond
-Migrate another double bond
Substrates used in -Stage 1 (HMG-CoA reductase) -Stage 2 -Stage 3 (squalene synthase) -Stage 4 (squalene monoxygnase: -Stage 1: 2 NADPH
-Stage 2: 3 ATP
-Stage 3: 1 NADPH
-Stage 4: 1 NADPH
3 Inherited disorders of Cholesterol Biosynthesis:: 1. Chondrodysplasia punctata
2. Latherosterolosis
3. Smith-lemli-Opitz syndrome
What are the cholest biosynth inherited disorders associated with?: Developmental malformities
What is the problem in these disorders?: Low cholesterol levels - lack of Hedgehog morphogens b/c they are made by post-transl. attachment of cholesterol.
What is the main regulatory target in cholesterol biosynth?: HMG-CoA Reductase
3 levels of Regulating HMG-CoA reductase:: 1. Gene Transcription
2. Proteolysis
3. Phosphorylation
How is gene transcription of HMG-CoA reductase regulated?: By SREBPs
Sterol Regulatory Element Binding Proteins
Where are SREBPs normally located? When is this the case?: In the ER - when cholesterol is high (biosynthesis unnecessary)
What happens to SREBPs when serum cholesterol gets low?: SCAP (on ER membrane) senses low levels; it travels to Golgi w/ SREBP, then cleaves SREBP
What part of SREBP gets cleaved; where does it go?: N-terminus -> goes to the nucleus to bind the SRE for HMG-CoA reductase.
What is SRE?: Sterol Regulatory Element
Result of SREBP binding to SRE?: Activated transcription of HMG-CoA reductase hence increased cholesterol biosynth.
What causes proteolysis of HMG-CoA reducatase?: metabolites of cholesterol
What 2 things do Cholesterol metabolites inhibit? What do they ACTIVATE?: -HMGCoA reductase (proteolysis)
-Extracellular uptake from LDL via receptor mediated endocytos.
-Activates ACAT for esterifictn
Phosphorylated HMG-CoA is ____ Dephosphorylated HMG-CoA is ____: Phosph = inactive

Dephosph = active
What stimulates HMG-CoA phosphorylation/dephosphoryltn?: Glucagon -> phosphorylate

Insulin -> dephosphorylate
How does glucagon stimulate phosphorylation of HMG-CoA red?: Via AMP-activated protein kinase
How does insulin stimulate dephosphorylation of HMG-CoA rd?: Via HMG-CoA Reductase Phosphatase
What are Statins?: Competitive Inhibitors of HMG-CoA reductase
What are the 4 Statins?: -Compactin
-Simvastatin (Zocor)
-Pravastatin (Pravachol)
-Lovastatin (Mevacor)
What are Statins used for?: To treat familial hypercholesterolemia
What pleiotropic effect is exhibited by statins?: Improved endothelial function via increased ENOS activity -
is how Viagra was discovered.
End product of cholesterol is:: Bile acids
Where are bile acids made? From what?: In liver from cholic acid
What is cholic acid?: Derivative of Cholesterol that is more soluble - 24 Carbons and 3 OH
What is the function of bile acids?: To emulsify fats in prep for pancreatic lipase
What happens to bile acids after release from gallbladder to intestine?: Reabsorbed - synthesis is not enough to meet physiolog demands.

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