Lectures 45 and 46
Problems associated with neonatal digestion
-Low pancreatic secretion of lipase
-Immature liver unable to produce bile
Overcome problems of neonatal digestion
- very active gastric lipase
- breast milk rich in MCFAs
- Breast milk also contains bile salt-stimulated lipase
Enzymes in De-novo Fatty acid synthesis
- ACCase (acetyl-CoA carboxylase)
- Fatty acid synthase (FAS)
Modifications of palmitate
Elongation
Desaturation
This takes place in the ER
**In mitochondria - Acyl oxidation, acetyl-CoA production, ketogenesis
**Cytosol - NADPH production, FA synthesis, isoprenoid synthesis
Simplified version of fatty acid synthesis
1. Acetyl CoA» Palmitate (enz = ACCase and FAS)
2. Palmitate » Unsaturated fat (desaturase) »
LCFA, VLCFA (elongase)
Palmitate » LCFA, VLCFA (elongase)
How are acetyl CoA made in the mitochondria transported to the cytosol for FA synthesis?
Malate-citrate shuttle
Malate-citrate shuttle
1. Acetyl-CoA + OAA » Citrate (TCA) (Citrate synthase)
2. Citrate crosses mitochondrial membrane » cytosol
3. Citrate » acetyl CoA + OAA (ATP-Citrate lyase)
Citrate + CoASH + ATP » Acetyl-CoA + OAA + ADP + Pi
(ATP- Citrate lyase) Cytosolic reaction
Why can FAs not be converted to carbohydrate in animals?
*** Acetyl CoA cannot be converted to pyruvate/ OAA
What happens to th OAA released into the cytosol from the citrate lyase reaction?
1. OAA + NADH » Malate +NAD (malate dehydrogenase) --- Malate diffuses through membrane into mitochondria
Also in cytosol
2. Malate + NADP » Pyruvate (Malic enzyme)
CArboxylation of Acetyl-CoA by ACCase in cytosol
1. Acetyl-CoA » Malonyl-CoA ( ACCase)
**ACCase is the only enzyme involved in FA synthesis that is separate from the multifunctional enzyme FAS (Fatty acid synthase)
Components of ACCase
- Biotin carboxylase
- transcarboxylase
-Biotin-carboxyl carrier protein ( BCCP)
Human ACCase
- multifunctional protein
- exists in inactive form
-active when it forms a filamentous polymer
**Rate of FA synthesis dependent on the eqm of those 2 forms:
Protomer (inactive) »« Polymer (active)
ACCase reation
2 phases:
1. Carboxylation of biotin to form carboxybiotin
(Biotin carboxylase)
ATP + HCO₃⁻ + BCCP » CO₂---BCCP + ADP + Pi
2. Transcarboxylation of biotin
(Transcarboxylase)
CO₂---BCCP + acetyl-CoA » malonyl-CoA + BCCP
Simplified FAS reaction
1. ACetyl CoA (2C) »Malonyl CoA (3C) (ACCase)
2. Malonyl CoA (3C) » Palmitic Acid (C16:0) (FAS using ACP intermediates)
3. Palmitic acid » Palmitoyl-CoA (thioester bond formation) *** increases the solubility.
Characteristics of FAS enzyme
- phosphopantetheine binding domain (ACP - acyl carrier protein)
- 2 thiol grps must be loaded onto acyl groups before condensation
- acyl grp from acyl-CoA initially transferred to ACP
- acyl grp then transferred from ACP to ketoacyl-ACP synthase
- malonyl grp from malonyl-CoA transferred to thiol grp of ACP
***ACP arm is flexible - move substrate to active site.
*** FAS has several active sites - 2 FAS work in pairs.
Preliminary reactions of FA synthesis
1. Acetyl-CoA + ACP » Acetyl-ACP + HS-CoA
2. Malonyl-CoA + ACP » Malonyl-ACP + HS-CoA
Condensation reactions of FA synthesis
acetyl synthase (2C) + malonyl-ACP(3C) » acetoacetyl-ACP(4C) + synthase-SH + CO₂(1C)
* enzyme - β-ketoacyl-ACP synthase.
1st Reduction reaction of FA synthesis
acetoacetyl-ACP + NADPH + H⁺ «» βhydroxybutyryl-ACP + NADP⁺
**Enz- βketoacyl-ACP reductase
2nd reduction reaction
crotonyl-ACP + NADPH + H⁺ »« butyryl-ACP (4C) + NADP⁺
enz- βenoyl-ACP reductase
Fate of butyryl-ACP
-enters 2nd round of FA synthesis similar to acetyl-ACP
*** Final product is PALMITOYL-ACP.
-palmitoyl-ACP » palmitic acid (thioesterase)
(All C's in palmitic acid are derived from malonyl-CoA except 2C's at methyl end (from original acetyl CoA molecule)
Effect of malonyl Co-A on CPT I
** inhibitory effect on CPT I
- prevents acetyl-CoA from β-oxidation
**Glucagon activates CPT I (liver)
Effect of insulin on FA synthesis
Insulin »» Acetyl CoA » Malonyl CoA » Pamitate
*Malonyl-CoA »»» inhibit CPT I»»»» no acetyl CoA goes into mitochondria»»» stays in cytosol for FA synthesis instead of oxidation
Elongation reactions of FA.
2 pathways:
1. ER (using malonyl-CoA)
2. Mitochondrial (uses acetyl-CoA)
Desaturation of FA reactions
-Aerobic
-2 H⁺ removed and H₂O produced
- NADPH used
**Acyl chain must be 16 or 18 C's before desaturation occurs.
Why can mammals not synthesize linoleic and α-linoleic acid?
- mammals cannot introduce Δ¹⁵ and Δ¹² double bonds.
-linoleate (C18:2) and α-linoleic (18:3) have this.
*** mammals can only synthesize up to Δ⁹ double bonds btwn the COOH and Δ⁹ of CH of chain.
**possible to desaturate oleate at Δ⁶ forming -
C18:2Δ⁶,⁹ FA.
Specific process : FA desaturation
Steroyl-CoA (C18:0) »»»oleoyl-CoA (C18:1 Δ⁹) + 2H₂O
*Aerobic desaturation
*microsomal enzymes
*Δ⁹ desaturation
Functions of essential FA's
- membrane structure
- specific enzyme-protein interaction in membranes
- synth eicosanoids
-syn arachidonic acid (C20:4)
-synth docosahexanoic acid (DHA C22:6)
PUFA
polyunsaturated FA
- not all are converted to eicosanoids bc of limiting activites of some enzymes (elongases and Δ⁵ and Δ⁶ desaturases)
- precursors for resolvins, docosatrienes, neuroprtectins (anti-inflammatory properties)
***** Humans rely on exogenous source of EPA and DHA in diet.
What is linoleic acid used to make?
- C18: 2n-6 (linoleic acid) » C18:3n-6 (γ linoleic acid) » C20:3n-6 » C20:3n-6» C20:4n-6 (Arachidonic acid) » C22: 5n-6
What is α-linolenic acid used to make?
-C18:3n-3 (α-linolenic acid) » C18:4n-3 » C20: 4n-3 » C20: 5n-3 (Eicosapentanoic acid, EPA) » C22: 6n-3 (Docosahexanoic acid, DHA)
When there is an EFA deficiency what happens?
Stearic acid is used to make Mead acid, causes dermatosis
Regulation of FA synthesis
-ACCase is key regulatory enzyme.
** strict regulation
1. Short term Rapid Response
2. Long term response
Short term rapid response FA regulation
ACCase
-allosterically (stimulated) regulated by citrate
-inhibited by palmitoyl-CoA
-regulated by phosphorylation/dephosphorylation
(insulin, epinephrine, norepinephrine, glucagon)
-Activated by insulin in dephosphorylated form,
-Deactivated by glucagon and epinephrine in phosphorylated form.
Long term Response
- Increased expression of ACCase and FAS (at molecular level to maintain high carb diet)
TAG synthesis - Kennedy pathway
- takes place in the liver
- exported to peripheral tissue in blood
- Glycerol-3-Phosphate is immediate precursor for TAG synthesis.
** Glycerol-3-P derived directly from LPLase or indirectly from glycolysis.
- acyl-CoA transferred from cytosolic acyl-CoA to SER
-(TAG molecule is assembled here)
Formation of phosphatidic acid
-2 sequential acylations
1. Glycerol-3-P acyl transferase,
Glycerol-3-P + Acyl-CoA»» lysophoshatidic acid
2. Acylglycerol-3-P acyltransferase
Lysophosphatidic acid+ Acyl-CoA»» Phosphatidic acid
3. Phosphate removed by phosphatase
Phosphatidic acid»» Diacylglycerol + Pi
4. DAG acyl transferase
Diacylglycerol + Acyl-CoA»» Triacylglycerol
Important Sites of TAG synthesis
-SI (MAG pathway)
-Liver ( Kennedy pathway)
-Adipose tissue (Kennedy)
-Mammary gland (milk fat lactation) (Kennedy pathway)
** small amount of turnover in myocytes.
-TAG synthesis in enterocytes is primarily controlled by the rate of influx of exogenous dietery lipids.
-TAG synthesis in adipose tissue governed by the supply of glucose-derived glycerol-3-P.
Important points to note about FA synthesis
1. De novo FA synthesis from glucose (ACCase and FAS) --- not very active in adipocytes due to Western diet
2. TAG synthesis utilizing glycerol-3-phosphate
3. TAG mobilization by HSL
4. FA uptake from TAG-rich blood lipoproteins following LPLase activity.
Cholesterol facts
- most common steroid in animal cells
- absent from plant tissue and vegetable oils
- OH makes it amphipatic
- cholesteryl esters are hydrophobic
- vital in cell membranes
- precursor to steroid hormones
-cannot be degraded to CO₂
- liver converts it to bile acids
Plant sterols
- plants synthesize sterols: stigmasterol, campesterol, β-sistosterol
- not readily absorbed by human intestines
- inhibitory effect on cholesterol absorption
- use in cholesterol lowering drugs
Difference btwn testosterone and estrogen
- Testosterone has an =O grp while estrogen has an OH grp.
Cholesterol synthesis simplified
*** in cytosol
-------acetyl-CoA + acetoacetl-CoA » HMG-CoA
1. HMGCoA » Mevalonate (HMG-CoA reductase)
2. Mevalonate » C5 Isopentyl PPi
3. C5 Isopentyl PPi » C10 Geranyl PPi
4.C10 Geranyl PPi » C15 Farnesyl PPi
5. C15 Farnesyl PPi » Squalene
6Squalene » Lanosterol » Cholesterol
** Farnesyl is precursor for
- CoQ (Ubiquinone)
- Dolichol (glycoprotein synthesis)