Fatty acid metabolism An overview TAG are highly concentrated energy source Steps in fa oxidation Additional steps are required for certain fas Fa synthesis Controlling fa metabolism Elongation and unsaturation of fas
Utilization of fas3 stages1. Mobilization ‒ TAG fa + glycerol2. Fa activation and transportation into mitochondria where oxidation takes place.3. Fa breakdown to Acetyl-CoA
FIGURE 17-3 Mobilization of triacylglycerols stored in adiposetissue. When low levels of glucose in the blood trigger the release ofglucagon, 1 the hormone binds its receptor in the adipocyte membraneand thus 2 stimulates adenylyl cyclase, via a G protein, to produce cAMP.This activates PKA, which phosphorylates 3 the hormone-sensitive lipaseand 4 perilipin (a family of proteins that restrict access to lipid droplets,preventing untimely lipid mobilization)molecules on the surface of thelipid droplet. Phosphorylation of perilipin permits hormone-sensitivelipase access to the surface of the lipid droplet, where 5 it hydrolyzestriacylglycerols to free fatty acids. 6 Fatty acids leave the adipocyte, bindserum albumin in the blood, and are carried in the blood; they arereleased from the albumin and 7 enter a myocyte via a specific fatty acidtransporter. 8 In the myocyte, fatty acids are oxidized to CO2, and theenergy of oxidation is conserved in ATP, which fuels muscle contractionand other energy-requiring metabolism in the myocyte.
Entry of electrons from fatty acid oxidation in the mitochondrial respiratory chain
Fatty Acid Oxidation In one pass through the fatty acid oxidation, one mol. Of Acetyl CoA, two pairs of electrons, and four hydrogen ions are removed. ─ CH3 - - - - COOH each time it is shorter by 2 C units The equation for one pass:(C16) palmitoyl-CoA + CoA + FAD + NAD + H2O myristoyl CoA + Acetyl-CoA + FADH2 + NADh + H+ Myristoyl CoA enters the -oxidation sequence, another set of four reactions, to give a second Acetyl-CoA.
Fatty Acid Oxidation Continued The overall reaction is:palmitoyl CoA + 7CoA + 7NAD + 7FAD + 7H2O 8 Acetyl CoA + 7 FADH2 + 7NADH + 7H+ These four steps are repeated (n/2 – 1) times for even numbered carbon chains. ─ FADH2 ETFP (electron transferring flavoprotein), provides 1.5 ATP ─ NADH complex I, provides 2.5ATP ─ Thus, four mol. of ATP are formed for each 2-C unit removed in one pass.
In hibernating animals, fatty acid oxidation provides energy, heat,and water. Fatty acid oxidation is also important for the camel. I) Palmitoyl-CoA + 7CoA + 7O2 + 28Pi + 28ADP 8Acetyl-CoA + 28 ATP + 7H2O II) Acetyl-CoA oxidized in the TCA cycle 1Acetyl-CoA 10ATP 8Acetyl-CoA 80ATP 8Acetyl-CoA + 16O2 + 80Pi + 80ADP 8 CoA + 80ATP + 16H2O + 16CO2Combine I and II Palmitoyl-CoA + 23O2 + 108Pi + 108ADP CoA + 108ATP + 16CO2 + 23H2O
Certain fatty acids require additional steps fordegradation Even numbered are fully saturated and are completely oxidized Not all fa are simple and even numbered The oxidation of fa containing double bonds require additional steps Odd numbered fa yield propionyl CoA at the final thiolysis step. We need to metabolize propionyl CoA.
Oxidation of unsaturated fatty acids This is somewhat difficult Two additional enzymes are required: • Isomerase • Reductase Let’s analyze the oxidation of palmitoleate • Activated • Transported • Undergoes 3 cycles of degradation by the same enzymes as in the oxidation of saturated fas. However, the cis-d3-enoyl CoA formed in the third round is not a substrate for acylCoA dehydrogenase. • This should be converted to trans by the ISOMERASE enzyme.
Another problem arises with the oxidation of pufas What if we have two double bonds? • Linoleate (18:9,12) Steps • Cis double bond is formed after 3 rounds of beta oxidation • This is coverted to a trans by ISOMERASE • The acylCoA produced by another round of beta oxidation contains a cis-delta 4 double bond. • Dehydrogenation of this yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the b-oxidation pathway. • This problem is solved by 2,4-DIENOYL CoA REDUCTASE! Thus, 2 extra enzymes are needed for the oxidation of even numbered pufas: isomerase and reductase
Oxidation of odd chain fatty acids Odd chain fas oxidation is the same except the last step. • The last step produces Acetyl CoA (2C) and Propionyl CoA (3C) How do we deal with 3C compound? • It is converted into succinyl CoA in a reaction that requires Vit B12. • Succinyl CoA is an intermediate in the TCA cycle.
Fas are also oxidized in peroxisomes Peroxisomes are membrane-enclosed cellular compartments. • Hydrogen peroxide is produced by fa oxidation and then destroyed enzymatically. • Peroxisomes have high levels of catalase. The process consists of 4 steps: 1) Dehydrogenation 2) Hydration 3) Oxidation 4) Thiolytic cleavage The differences: • In peroxisomes, the flavoprotein dehydrogenase passes electrons directly to oxygen. • The NADH formed in peroxisomes cannot be reoxidized, and the peroxisome must export reducing equivalents to the cytosol.
Peroxisomal degradation Fa oxidation in these organelles stop at octanyl CoA • Meaning, peroxisomes serve to shorten long chains to make them better substrates for beta oxidation in mitochondria. Zelweger syndrome, which results from the absence of functional peroxisomes, is characterized by liver, kidney, and muscle abnormalities and usually results in death by age 6.
Ketone bodies are formed from Acetyl CoA when fat breakdown predominates Acetyl CoA enters the TCA cycle only if fat and ch degradation are balanced. If oxaloacetate is decreased (if ch is unavailable) then acetyl CoA will not enter TCA cycle. Also, in fasting and diabetes, oxalocetate is consumed to make glc by the gluconeogenic pathway; therefore, acetyl CoA increases. Under these conditions increased acetyl CoA makes KETON BODIES • Acetoacetate • Beta hydroxybutyrate • Acetone
Utilization of ketone bodiesby extrahepatic tissues
Fatty Acid Synthesis Production of cytoplasmic Acetyl-CoA Carboxylation of Acetyl-CoA to form malonyl-CoA Fatty acid synthesis by a multi-enzyme complex Regulation of fatty acid synthesis Metabolism of unsaturated fatty acids and eicosanoids
Fatty Acid Synthesis It is not a reversal of fa oxidation. It occurs in the cytoplasm of the cells of the liver, fat tissue, and mammary gland and, to a lesser extent, in the cytoplasm of the cells of the kidney. The process incorporates carbons from Acetyl CoA (the immediate substrate) into growing a fa chain, using ATP and NADPH as cofactors. Fa synthesis is increased by insulin. Production of cytoplasmic acetyl CoA: • Transfer of acetate units from mitochondrial acetylCoA to cytoplasm, forming cytoplasmic acetyl CoA, since CoA can not cross the mitochondrial membrane. • Carboxylation of AcetylCoA to form malonyl CoA • The irreversible formation of malonyl CoA from acetylCoA is catalyzed by acetylCoA carboxylase. Biotin, prosthetic group (The 2 step reaction, similar to other biotin-dependent carboxylation reactions. Others: Pyruvate carboxylase, propionylCoA carboxylase) • The CO2 derived from HCO3 is first transferred to biotin in an ATP-dependent reaction. The biotinyl group serves as a temporary carrier of CO2.
Citrate carries acetyl groups frommitochondria to the cytosol for fa synthesis The synthesis of palmitate requires 8 mols of acetyl CoA, 14 mols of NADPH, and 7 mols of ATP. Fa synthesis is in the cytosol. Acetyl CoA is made from pyruvate in the mitochondria. So, Acetyl CoA must be transferred from the mitochondria to the cytosol. Solution: acetyl groups are carried as citrate. • When citrate is high, it is transported to the cytosol. In the cytosol, it is cleaved by ATP-citrate lyase.
Carboxylation of acetyl CoA to form malonyl CoAby Acetyl-CoA carboxylase Acetyl-CoA carboxylase has 3 functional subunits: 1) Biotin carrier protein. 2) Biotin carboxylase, • which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction. 3) Transcarboxylase • which transfers activated CO2 from biotin to acetyl-CoA producing malonyl CoA. The long-flexible biotin arm makes this.
The formation of Malonyl-CoA The formation of Malonyl-CoA is the committed step in fa synthesis. Fa synthesis starts with carboxylation of acetyl-CoA to malonyl-CoA. • Irreversible reaction • Biotin is a cofactor • ATP is also required.
Important facts about the synthesis:1) Synthesis takes place in the cytosol; oxidation, in the mitochondria.2) Intermediates in fa synthesis are covalently linked to the ACP (acyl carrier protein), whereas in oxidation, they are bonded to CoA.3) Fa Synthesis enzymes are joined in a single pp chain called fa synthase. Oxidation is not like that.4) Growing of the chain is done by adding 2C units obtained from Acetyl-CoA. Activated donor of 2C units is Malonyl-CoA.5) The reductant in fa synthesis is NADPH.6) Elongation stops when C16 is made.
Fatty Acid SynthesisThe remaining series of reactions are catalyzed by amultienzyme complex.• In eukaryotes this enzyme complex, fatty acid synthase, consists of 2 subunits that, together, have 7 enzymatic activities.• They are polypeptides.• The proteins act together to catalyze the formation of fatty acids from acetyl coA and malonyl CoA.• The fundamental reaction order by which the long chains of carbon atoms in fas are assembled consists of 4 steps. 1) Condensation of Acetyl group with malonyl group. 2) Reduction of the Carbonyl Group 3) Dehydration 4) Reduction of the Double Bond
Fatty Acid Synthesis This is one passage. With each passage through the cycle, the fatty acyl chain is extended by 2 carbons. When the chain length is 16, the product (palmitate 16:0) leaves the cycle. The fa synthase complex has 7 different active sites polypeptides. • Throughout the process, the intermediates remain covalently attached to one of two thiol groups of the complex. • One point of attachment is the -SH group of a Cys residue in one of the seven proteins. • Beta-ketoacyl ACP the other is the -SH group of acyl carrier protein (ACP). ACP is a small protein (Mr 8860) containing the prosthetic group 4 phosphopantetheine, an intermediate in the synthesis of coenzyme A.
Fatty Acid Synthesis Biosynthesis of fa is a 4-step sequence that lengthens a growing f. acyl chain by two carbons. 1) Condensation • CO2 is eliminated from malonyl group. • Net effect is extension of the acyl chain by two carbons. • The beta group is then reduced in three more steps nearly identical to the reactions of beta oxidation, but in the reverse sequence. 2) The beta-keto group is reduced to an alcohol. 3) The elimination of H2O creates a double bond 4) The double bond is reduced to form the corresponding saturated f.acyl group. The fa chain grows by two-carbon units that are donated by activated malonate, with loss of CO2. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of 4, then 6, 8, and so on. The final product is palmitate (16:0).
Intermediates in fa synthesisare attached to ACP Intermediates are linked to ACP • Specifically, to the -SH terminus of a phosphopantetheine group which is linked to a Ser residue of ACP ACP is a single polypeptide chain of 77 residues. ACP can be regarded as a giant prosthetic group (macro CoA). Both ACP and CoA include phosphopantetheine as their reactive units
Fa synthase inhibitors Fa synthase is overexpressed in some breast cancers. Some inhibitors were tested on mice and a great weight loss was observed.Therefore, fa synthase inhibitors are exciting candidates,both as anti-tumor and as anti-obesity drugs!
Fa biosynthesis requirements The biosynthesis of fa require Acetyl CoA, ATP and NADPH. • The ATP is required to attach CO2 to acetylCoA to make malonyl CoA • NADPH is required to reduce the double bonds. 1. NADPH supplied from HMP or cytoplasmic conversion of malate to pyruvate. 2. Malate is oxidized and decarboxylated by a cytoplasmic NADP-dependent malate dehydrogenase or malic enzyme to form pyruvate.
Fatty acid biosynthesis is tightly regulated If we have more energy than our needs, fatty acids are stored. Acetyl coA carboxylase is the rate limiting step in the biosynthesis of fatty acids (Important site regulation). The reaction is catalyzed by acetyl coA carboxylase • Switched off by phosphorylation • Activated by dephosphorylation The carboxylase is regulated by 3 signals: 1. Glucagon – inhibits carboxylase activity 2. Epi – inhibits carboxylase activity 3. Insulin – stimulates fas synthesis Control is also done by the levels, within the cell, of 1. Citrate – activates carboxylase • Energy and building blocks are abundant, and we can go ahead and store them 2. Palmitoyl CoA – inhibits carboxylase 3. AMP – inhibits carboxylase
More regulation The proportion of active carboxylase depends on the catalytic rates of these opposing enzymes. • Protein kinase A inhibits phosphatase by phosphorylating it. - Carboxylase stays in its inactive form. • The inactive form also predominates when the energy level of the cell is low. - Phosphorylation is stimulated by high AMP levels. • Insulin stimulates carboxylase perhaps by activating protein phosphatase 2A. How about citrate’s role? • Citrate is high when acetyl CoA and ATP are high. • When citrate is high and ATP is available, we can start making fa. • Citrate stimulates carboxylase. • Palmitoyl CoA inhibits this action of citrate on the carboxylase enzyme.
Dependence of the catalytic activity of acetyl CoA carboxylase on the concentration of citrate No citrate: Dephosphorylated form is predominant The presence of citrate partly reverses the inhibition produced by phosphorylation for the enzyme (Acetyl CoA carboxylase) Citrate facilitates the polymerization of the inactive octamers into active filaments • Acetyl CoA carboxylase exists as an octamer The level of citrate is high when acetyl CoA and ATP are abundant. Hence, increased level of citrate signifies that 2-C units and ATP are available for the fa synthesis
More regulation The stimulatory effect of citrate on the carboxylase is antagonized by palmitoyl CoA, which is abundant when there is an excess of fatty acids Palmitoyl CoA causes the filaments to disassemble into the inactive octamers Response to diet: In starvation, ffa are increased because Epi, Glucagon stimulate lipase. Insulin, in contrast, inhibits lipolysis Also, malonyl CoA inhibits carnitine shuttle preventing excess of fatty acyl CoAs to the mitochondrial matrix in times plenty.
Elongation and unsaturation The major product of fa synthesis is palmitate Longer ones are formed by elongation reactions in ER 2C units are added, the donor is still malonyl CoA ER enzymes also introduce double bonds.There is an electron-transport chain in the desaturation offatty acids which includes NADH-cytochrome b5-reductase,cytb5, and desaturase.
pufas Some pufas can not be synthesized by mammals and are nutritionally essential • C20, 22, and 24 fa may be detected in the tissues. They are derived from oleic (in plants but not animals) and linoleic acid by chain elongation. • Palmitoleic and oleic acids are not essential in the diet because animals can make a double bond at the 9 position. • Linoleic and linolenic acids are essential fas because animals cannot synthesize them. • Arachidonic acid can be formed from linoleic acid in most animals. In animals, double bonds can be introduced at the 4,5,6 and 9 positions but never beyond the 9 position. Plants can introduce double bonds beyond 9 position and can, therefore, synthesize essential fas.
More about pufas Monounsaturated fatty acids are synthesized by a desaturase system. Many tissues, including liver tissues, can make monounsaturated forms. The first double bond introduced into a saturated fa is almost always at the 9 position. Enzyme is desaturase. Synthesis of pufas involves desaturase and elongase systems. In animals, the additional double bonds all introduced between the existing double bond and the -COOH group, but in plants, they may also be introduced between the 9 and omega carbon. • Linoleate 18:2(9,12) and linolenate 18:3(9,12,15) cannot be synthesized by mammals, but plants can synthesize both. • The plant desaturases that introduce double bonds at 12 and 15 positions are located in SER.
Eicosanoids Eicosanoids are formed from C20 pufas TXA, LT and PGs are called eicosanoids. Arachidonate gives rise to Pgs, TX and LT. • A family of very potent biological molecules are made from arachidonate. • They act as short-range messengers, affecting neighboring tissues. • In response to a hormonal or other stimulus, a specific phospholipase affects membrane phospholipids, releasing arachidonate. SER enzymes then convert arachidonate into prostaglandins, beginning with the formation of PGG2, very first PGs