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陳泰源 博士

taid@gate.sinica.edu.tw

中央研究院 生物化學研究所
2007/5/29 National Taiwan Normal University

Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration
• Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl CoA. • Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted • Stage 3: Electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial electron carriers--the respiratory chain--- reducing to O2 to H2O--- production of ATP

Good and bad of using triacylglycerols as fuel storage
• The long alkyl chains of their constituent fatty acids are essentially hydrocarbons, highly reduced structures with an energy of complete oxidation (~38 kJ/g) more than twice that for the same weight of carbohydrate or protein. • extreme insolubility of lipids in water; cellular triacylglycerols aggregate in lipid droplets, which do not raise the osmolarity of the cytosol, and they are unsolvated. (In storage polysaccharides, by contrast, water of solvation can account for two-thirds of the overall weight of the stored molecules.) And because of their relative chemical inertness, triacylglycerols can be stored in large quantity in cells without the risk of undesired chemical reactions with other cellular constituents. • Because they are insoluble in water, ingested triacylglycerols must be emulsified (乳化) before they can be digested by water-soluble enzymes in the intestine, and triacylglycerols absorbed in the intestine or mobilized from storage tissues must be carried in the blood bound to proteins that counteract their insolubility. • To overcome the relative stability of the C-C bonds in a fatty acid, the carboxyl group at C-1 is activated by attachment to coenzyme A, which allows stepwise oxidation of the fatty acyl group at the C-3, or b position— hence the name b oxidation.

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with head groups facing the aqueous phase. Several apolipoproteins that protrude from the surface (B-48. The diameter of chylomicrons ranges from about 100 to 500 nm. HDL. CII) act as signals in the uptake and metabolism of chylomicron contents. C-III. (2). • Apolipoproteins are lipid-binding proteins in the blood. stored in cells as lipid droplets. (3). combine with lipids to form several classes of lipoprotein particles.Molecular structure of a chylomicron • Cells can obtain fatty acid fuels from three sources: (1). synthesized in one organ for export to another. Triacylglycerols sequestered in the interior (yellow) make up more than 80% of the mass. LDL. fats consumed in the diet. . • The surface is a layer of phospholipids. spherical aggregates with hydrophobic lipids at the core and hydrophilic protein side chains and lipid head groups at the surface: chylomicrons and very low density lipoproteins (VLDL).

2. 3. 7. In the myocyte.[glucose]↓=> Epinephrine glucagon Hormones Trigger Mobilization of Stored Triacylglycerols • 1. lipid droplets in adipocytes (and in steroid synthesizing cells of the adrenal cortex. fatty acids are oxidized to CO2. 4. . 5. to produce cAMP. Phosphorylation of perilipin permits hormone sensitive lipase access to the surface of the lipid droplet it hydrolyzes triacylglycerols to free fatty acids (FFA). and testes) is coated with perilipins. which fuels muscle contraction and other energy requiring metabolism in the myocyte. the hormone binds its receptor in the adipocyte membrane stimulates adenylyl cyclase. they are released from the albumin and enter a myocyte via a specific fatty acid transporter. via a G protein. ovary. FAs leave the adipocyte. When low levels of glucose in the blood trigger the release of glucagon. 6. 8. and the energy of oxidation is conserved in ATP. This activates PKA PKA phosphorylates the hormone-sensitive lipase PKA phosphorylates perilipin molecules on the surface of the lipid droplet. bind serum albumin in the blood. preventing lipid mobilization. and are carried in the blood.

. and the resulting glycerol 3phosphate is oxidized to dihydroxyacetone phosphate.Fate of Glycerol • About 95% of the biologically available energy of triacylglycerols (TGs) resides in their three longchain fatty acids. The glycolytic enzyme triose phosphate isomerase converts this compound to glyceraldehyde 3-phosphate. • The glycerol released by lipase action is phosphorylated by glycerol kinase. only 5% is contributed by the glycerol moiety. which is oxidized via glycolysis.

the pyrophosphate formed in the activation reaction is immediately hydrolyzed by inorganic pyrophosphatase. which pulls the preceding activation reaction in the direction of fatty acyl–CoA formation. Fatty acyl–CoAs are high-energy compounds. coupled to the cleavage of ATP to AMP and PPi. their hydrolysis to FFA and CoA has a large.Conversion of a fatty acid to a fatty acyl–CoA outer mitochondrial membrane • acyl-CoA synthetases (outer mitochondrial membrane) catalyze the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA. • . The formation of a fatty acyl–CoA is made more favorable by the hydrolysis of two highenergy bonds in ATP. negative standard free-energy change.

the carnitine ester is formed on the cytosolic face of the outer membrane. fatty acids. (2). then moved across the outer membrane to the intermembrane space through porin. This transesterification is catalyzed by carnitine acyltransferase I (1). the acyl-CoA passes through the outer membrane and is converted to the carnitine ester in the intermembrane space. and some amino acids  whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids .Fatty Acids Are Activated and Transported into Mitochondria (size dependent) • • • • The fatty acids with chain lengths < = 12C without transporters  >=14C use carnitine shuttle to enter mitochondria: carnitine acyltransferase is inhibit by [malonyl-CoA] transiently attached to the hydroxyl group of carnitine to form fatty acyl–carnitine—the second reaction of the shuttle. This isozyme. in the outer membrane. located on the inner face of the inner mitochondrial membrane. regenerates fatty acyl–CoA and releases Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate. – then facilitated diffusion through the acyl-carnitine/carnitine transporter fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II.

2.g. At the end of seven cycles the last two carbons of palmitate (originally C-15 and C-16) remain as acetylCoA => eight two-carbon acetyl groups of acetyl-CoA molecules. in each pass losing two carbons as acetyl-CoA. e. fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA. donate electrons to the mitochondrial respiratory chain.Stages of fatty acid oxidation • mitochondrial oxidation of fatty acids takes place in three stages: 1. Formation of each acetyl-CoA requires removal of four hydrogen atoms (two pairs of electrons and four H) from the fatty acyl moiety by dehydrogenases. starting from the carboxyl end of the fatty acyl chain. acetyl-CoA are oxidized to CO2 in the citric acid cycle (matrix). through which the electrons pass to oxygen with the concomitant phosphorylation of ADP to ATP . the 16-carbon palmitic acid undergoes seven passes through the oxidative sequence. 3. the reduced electron carriers NADH and FADH2.

thiolysis: acyl-CoA acetyltransferase (thiolase) promotes reaction of . acetyl-CoA feedback inhibit thiolase Trifunctional protein (TFP) 2.VLCAD (12-18C) 2. each specific for a range of fatty-acyl chain lengths: very-long-chain acyl-CoA dehydrogenase: (1). MCAD . The other product is the coenzyme A thioester of the fatty acid. SCAD (4-8C) The Oxidation of Saturated Fatty Acids Has Four Basic Steps 1. now shortened by two carbon atoms. [acetyl-CoA] ↑ . inhibit when [NADH]/[NAD]↑ ratio 4. 3.NAD (inhibit by high [NADH]/[NAD] ratio). (2). enoyl-CoA hydratase: water is added to the double bond of the trans-2-enoyl-CoA to form the L stereoisomer of b-hydroxyacyl-CoA L-b-hydroxyacyl-CoA is dehydrogenated to form -ketoacyl-CoA. Very-Long-Chain AcylCoA Dehydrogenase (VLCAD) .12 to 18C. MCAD (4-14C) 3.1. by the action of – bhydroxyacyl-CoA dehydrogenase .4 to 8C.ketoacylCoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetylCoA.4 to 14C. SCAD . three isozymes of acyl-CoA dehydrogenaseFAD. (3).

5th ed) .(Stryer’s Biochem.

see also in Table 17. with the enzymes employed depending on the length of the fatty acyl chain.5 X 7) = 28 ATP 8 acetyl-CoA  TCA ETC (10 X 8) = 80 ATP sum = 108 ATP.7 run 7 FADH2 (1. Each subunit contains two activities. PP. the enoyl-CoA hydratase and the b-hydroxyacyl-CoA dehydrogenase. the subunits contain the thiolase activity. For fatty acyl chains of 12 or more carbons.• substrate channeling: The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes. the reactions are catalyzed by trifunctional protein (TFP in inner mitochondrial membrane) heterooctamer of a4b4 subunits.5 X 7) + 7 NADH (2. 640 .1. one run of b-oxidation b-oxidation of palmitoyl-CoA (16C) .

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heat. • Urea formed during breakdown of amino acids is reabsorbed in the kidneys and recycled.Fat Bears Carry Out b Oxidation in Their Sleep • Transfer of electrons from NADH or FADH2 to O2 yields one H2O per electron pair. • In hibernating animals. • The glycerol released by degradation of triacylglycerols is converted into blood glucose by gluconeogenesis. the amino groups reused to make new amino acids for maintaining body proteins. . fatty acid oxidation provides metabolic energy. and water — all essential for survival of an animal that neither eats nor drinks for long periods.

nine acetylCoAs are produced from one molecule of the 18-carbon oleate. decanoyl-CoA. Altogether. which is converted by enoylCoA hydratase into the corresponding L-hydroxyacylCoA (trans-2-dodecenoyl-CoA). • This intermediate is now acted upon by the remaining enzymes of oxidation to yield acetylCoA and the coenzyme A ester of a 10. The latter undergoes four more passes through the pathway to yield five more molecules of acetylCoA. D enoyl-CoA isomerase isomerizes the cis-3-enoyl-CoA to the trans-2-enoyl-CoA.Oxidation of a monounsaturated fatty acid -Two Additional Reactions 3 2 • The auxiliary enzyme D .carbon saturated fatty acid. .

Oxidation of a polyunsaturated fatty acid • the combined action of enoyl-CoA isomerase and 2.4-dienoyl-CoA reductase. convert unsaturated fatty acid with a cis-3. .cis-6 configuration intermediate into regular b oxidation.

1. L-methylmalonyl-CoA to succinyl-CoA by methylamlonyl-CoA epimerase • succinyl-CoA enter TCA 1 2 TCA 3 .Complete Oxidation of Odd-Number Fatty Acids . carboxylation of propionyl-CoA to D-methylmalonyl-CoA by propionyl-CoA carboxylase 2.Requires Three Extra Reactions • Cattle and other ruminant animals form large amounts of the three carbon propionate (CH3-CH2-COO-). convert D-methymalonyl-CoA to L-methylmalonyl-CoA by methylmalonyl-CoA epimerase 3.

formed by cleavage of the triphosphate of ATP.dimethylbenzimidazole ribonucleotide bound covalently by its 3-phosphate group to a side chain of the corrin ring. A fifth coordination position . • can be synthesized only by a few species of microorganisms (3 mg/day is enough) • pernicious anemia 惡性貧血 (lack of RBCs. Hb. through aminoisopropanol. impair CNS) result form missing a glycoprotein which is required form B12 absorption. is coordinated with heme and.Coenzyme B12 • exchange of an alkyl or substituted alkyl group (X) with a hydrogen atom on an adjacent carbon • Cobalt (Co3+) complexed with corrin ring system. .

sleepiness. low blood glucose (hypoglycemia). high blood levels of octanoic acid. • One of the most severe disorders results from loss of the longchain -hydroxyacyl-CoA dehydrogenase activity of the trifunctional protein. Other disorders include defects in the or subunits that affect all three activities of TFP and cause serious heart disease and abnormal skeletal muscle. The pattern of organic acids in the urine helps in the diagnosis of this disease: the urine commonly contains high levels of 6-carbon to 10-carbon dicarboxylic acids (produced by w oxidation) and low levels of urinary ketone bodies • low-fat. high carbohydrate diet and avoiding long intervals between meals help when detected early. and northern European populations is due to a mutation in the gene encoding the medium-chain acyl-CoA dehydrogenase (MCAD) unable to oxidize fatty acids of 6 to 12 carbons • fat accumulation in the liver. . and coma. vomiting. TFP.Genetic Defects in Fatty Acyl–CoA Dehydrogenases Cause Serious Disease • The most common genetic defect in fatty acid catabolism in U.S.

malonyl-CoA inhibits carnitine acyltransferase I. PKA phosphorylates and inactivates ACC. 4. glucagon also triggers the mobilization of fatty acids in adipose tissue (ref. . [glucose]blood ↑ triggers the release of insulin. 8. 3. 5 . Insulin-dependent protein phosphatase dephosphorylates and activates ACC. FA can not enter mitochondrial matrix. 17-3). [malonyl-CoA]↓. 2. 6.glucagon release  activates cAMP-dependent protein kinase (PKA). FAs enter the mitochondrial matrix. 7. to Fig. 1.Coordinated regulation of fatty acid synthesis and breakdown • • • • the rate of transfer of long-chain fatty acyl–CoA into mitochondria: FA to TG in cytosol or b oxidation in mitochondria. ACC catalyzes the formation of malonyl-CoA. b oxidation. [glucose]blood ↓.

generating H2O2. peroxisomes fail to import long chain FA in peroxisomes In mammals. X-linked adrenoleukodystrophy (XALD. high concentrations of fats in the diet result in increased synthesis of the enzymes of peroxisomal b oxidation in the liver. Liver peroxisomes do not contain the enzymes of the citric acid cycle => long-chain or branched fatty acids are catabolized to shorter-chain products (hexanoyl-CoA). lack of a functional transporter). (2) in mammals the peroxisomal system is much more active on very-long chain fatty acids such as hexacosanoic acid (26:0) and on branched-chain fatty acids such as phytanic acid and pristanic acid Zellweger syndrome: unable to make peroxisomes and therefore lack all the metabolism unique to that organelle. which are exported to mitochondria and completely oxidized.  catalase (heat) and the NADH formed in the second oxidative step are exported to the cytosol. the major site of b oxidation is not mitochondria but peroxisomes.Comparison of oxidation in mitochondria and in peroxisomes/glyoxysomes • catalase • Heat • • To cytosol then to mitochondria • • In plant cells. two differences : (1) in plant: the first oxidative step electrons pass directly to O2. eventually entering mitochondria. during seed germination: acetyl-CoA is converted via the glyoxylate cycle to four-carbon precursors for gluconeogenesis .

and a wide variety of essential metabolites. which converts the H2O2 produced by oxidation to H2O and O2 . sucrose. contain high concentrations of catalase.Triacylglycerols as glucose source in seeds • In plants. • Glyoxysomes like peroxisomes. Fatty acids released from the triacylglycerols are first activated to their coenzyme A derivatives and oxidized in glyoxysomes by the same four-step process that takes place in peroxisomes • The acetyl-CoA produced is converted via the glyoxylate cycle to four-carbon precursors for gluconeogenesis. fatty acid oxidation does not occur primarily in mitochondria (as noted earlier) but in the peroxisomes of leaf tissue and in the glyoxysomes of germinating seeds (similar in structure). • During seed germination. stored triacylglycerols are converted into glucose.

. one of which contains four enzymatic activities in a single polypeptide chain: The first enzyme. acyl-CoA oxidase. however. soluble enzyme peroxisomes and glyoxysomes (Gram negative): long chain FA. membrane-associated enzyme • The b-oxidation enzymes of plant peroxisomes and glyoxysomes. form a complex of proteins. is a single polypeptide chain. Enz5: D-3-hydroxyacyl-CoA epimerase Enz6: D3. Enz3: L-b-hydroxyacyl-CoA dehydrogenase Enz4: thiolase. the multifunctional protein (MFP) contains the second and third enzyme activities (enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase) as well as two auxiliary activities needed for the oxidation of unsaturated FA Enz1: acyl-CoA dehydrogenase. Enz2: enoyl-CoA hydratase. D2-enoyl-CoA isomerase.The b-Oxidation Enzymes of Different Organelles Have Diverged during Evolution • Mitochondria (Gram positive): short chain FA.

alcohol dehydrogenase oxidizes the hydroxyl group to an aldehyde. producing a fatty acid with two carboxyl group • both ends can be attached to coenzyme A . aldehyde dehydrogenase oxidizes the aldehyde group to a carboxylic acid. in the ER of liver and kidney.most distant from the carboxyl group (10-12C). introduces a hydroxyl group onto the carbon by mixed function oxidases.TCA . 1. 3. 2.The w oxidation of fatty acids in the ER  w carbon .

resulting from a genetic defect in phytanoyl-CoA hydroxylase. which now has no substituent on the b carbon and can be oxidized further by b oxidation. leads to very high blood levels of phytanic acid and severe neurological problems including blindness and deafness. and then oxidized to the corresponding carboxylic acid.The a oxidation of a branchedchain fatty acid (phytanic acid) in peroxisomes • phytanoyl. decarboxylated to form an aldehyde one carbon shorter. in a reaction that involves molecular oxygen. • Refsum’s disease.CoA is hydroxylated on its a carbon. .

converted to acetyl-CoA and oxidized in TCA – provide energy for skeletal and heart muscle and the renal cortex. The brain. which preferentially uses glucose as fuel. when glucose is unavailable.Ketone Bodies • acetyl-CoA in the liver during oxidation of fatty acids can either enter the TCA or covert to the ―ketone bodies‖ • Acetoacetate and D-b-hydroxybutyrate are transported by the blood to tissues other than the liver (extrahepatic tissues) . .g. e. can adapt to the use of acetoacetate or D-b-hydroxybutyrate under starvation conditions.

GTP and. NADH. ultimately. ATP. but no CHO present – Starvation – Forms ketone bodies (see fat metabolism slides) – Danger! . NADH. ATP • If energy being used.Fates of Acetyl CoA O TAG's H3C SCoA Kreb's CO2. FADH2.energy acetyl CoA no CHO present ketone bodies • • In the presence of CHO and using energy If energy not being used (Lots of ATP present) – Made into fat – Metabolized to CO2...

• The six-carbon compound HMG-CoA is also an intermediate of sterol biosynthesis. thiolase catalyzes the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. (in the matrix of liver mitochondria). When acetyl-CoA accumulates (as in starvation or untreated diabetes) 1. Formed in the Liver. well-nourished individuals produce ketone bodies at a relatively low rate. Are Exported to Other Organs as Fuel • Healthy. cleaved by HMG-CoA lyase to acetoacetate and acetyl-CoA.Ketone Bodies. 2. sterol biosynthesis (cytosol) mitochondrial matrix . acetoacetyl-CoA then condenses with acetyl-CoA to b-hydroxy-bmethylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase 3. but the enzyme that forms HMG-CoA in that pathway is cytosolic.

The acetoacetate is activated to its coenzyme A ester by transfer of CoA from succinyl.D-b-Hydroxybutyrate as a fuel • D-b-hydroxybutyrate is oxidized to acetoacetate by Db-hydroxybutyrate dehydrogenase. which enter the citric acid cycle. • in a reaction catalyzed by ketoacyl-CoA transferase. The acetoacetyl-CoA is then cleaved by thiolase to yield two acetyl-CoAs. Thus the ketone bodies are used as fuels .CoA. an intermediate of the citric acid cycle.

Ketone Bodies Are Overproduced in Diabetes and during Starvation • • During starvation.can not inhibit carnitine acyltransferase I .extrahepatic tissues cannot take up glucose efficiently from the blood. Under these conditions. diverting acetyl-CoA to ketone body production In untreated diabetes. also have increased levels of ketone bodies in their blood and urine. [ insulin]↓ .fatty acids enter mitochondria to be degraded to acetyl-CoA— which cannot pass through the citric acid cycle because cycle intermediates have been drawn off for use as substrates in gluconeogenesis.000 mg/24 hr (compared with a normal rate of 125 mg/ 24 hr). [malonyl-CoA] ↓ .extrahepatic tissues can not fully oxidize them => [acetoacetate and D-b-hydroxybutyrate] ↑↑↑ . • • ketosis酮體中毒 : 90 mg/100 mL (compared with a normal level of 3 mg/100 mL) and urinary excretion of 5. These levels must be monitored to avoid the dangers of acidosis and ketosis (ketoacidosis酮症酸中毒 ). [acetylCoA]↑↑↑. gluconeogenesis depletes citric acid cycle intermediates.lower the blood pH => acidosis酸中毒 coma and death. either for fuel or for conversion to fat. Individuals on very low-calorie diets. . using the fats stored in adipose tissue as their major energy source.