Question

The process of β-oxidation requires fatty acids to be activated by transfer to Coenzyme- A. This step is dependent on free energy provided by ATP.

Briefly describe how breakage of high-energy bonds in ATP is linked to the transfer of fatty acids to Coenzyme A. What critical fatty acid intermediate is formed in this process? How is energy of phosphoanhydride bonds in ATP leveraged to facilitate the formation of the thioester bond between the fatty acid and Coenzyme A moieties? Draw structures as needed to clarify your points.

2) Explain the four-step process of the fatty acid β-oxidation pathway starting with palmitoyl-CoA. Provide reactions with structures as well as names of reactants, products, cofactors and enzymes.

Answer 1

β Oxidation

Mitochondrial oxidation of fatty acids takes place in three stages (Fig. 16-7). In the first stage-β oxidation-the fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acyl chain. For example, the 16-carbon fatty acid palmitic acid (palmitate at pH 7) undergoes seven passes through this oxidative sequence, in each pass losing two carbons as acetyl-CoA. At the end of seven cycles the last two carbons of palmitate (originally C-15 and C-16) are left as acetyl-CoA. The overall result is the conversion of the 16-carbon chain of palmitate to eight two-carbon acetyl-CoA molecules. Formation of each molecule of acetyl-CoA requires removal of four hydrogen atoms (two pairs of electrons and four H+) from the fatty acyl moiety by the action of dehydrogenases. In the second stage of fatty acid oxidation the acetyl residues of acetyl-CoA are oxidized to CO2 via the citric acid cycle, which also takes place in the mitochondrial matrix. Acetyl-CoA derived from fatty acid oxidation thus enters a final common pathway of oxidation along with acetyl-CoA derived from glucose via glycolysis and pyruvate oxidation (see Fig. 15-1). The first two stages of fatty acid oxidation produce the reduced electron carriers NADH and FADH2, which in the third stage donate electrons to the mitochondrial respiratory chain, through which the electrons are carried to oxygen (Fig. 16-7). Coupled to this flow of electrons is the phosphorylation of ADP to ATP, to be described in Chapter 18. Thus energy released by fatty acid oxidation is conserved as ATP. We will now look in more detail at the first stage of fatty acid oxidation, for the simple case of a saturated chain with an even number of carbons, and for the slightly more complicated cases of unsaturated and odd-number chains. We then consider the regulation of fatty acid oxidation, and the β-oxidative processes occurring in organelles other than mitochondria. In the second step of the fatty acid oxidation cycle (Fig. 16-8a), water is added to the double bond of the trans-Δ2-enoyl-CoA to form the L stereoisomer of β-hydroxyacyl-CoA (also designated β-hydroxyacyl-CoA). This reaction, catalyzed by enoyl-CoA hydratase, is formally analogous to the fumarase reaction in the citric acid cycle, in which H2O adds across an α-β double bond (p. 458). In the third step, the L-β-hydroxyacyl-CoA is dehydrogenated to form β-ketoacyl-CoA by the action of β-hydroxyacyl-CoA dehydrogenase (Fig. 16-8a); NAD+ is the electron acceptor. This enzyme is absolutely specific for the r. stereoisomer. The NADH formed in this reaction donates its electrons to NADH dehydrogenase (Complex I), an electron carrier of the respiratory chain (Fig. 16-9). Three ATP molecules are generated from ADP per pair of electrons passing from NADH to O2 via the respiratory chain. The reaction catalyzed by β-hydroxyacyl-CoA dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric acid cycle (p. 459). The fourth and last step of the fatty acid oxidation cycle is catalyzed by acyl-CoA acetyltransferase (more commonly called thiolase), which promotes reaction of β-ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the coenzyme A thioester of the original fatty acid, now shortened by two carbon atoms (Fig. 16-8a). This reaction is called thiolysis, by analogy with the process of hydrolysis, because the β-ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A. The Four Steps Are Repeated to Yield Acetyl-CoA and ATP In one pass through the fatty acid oxidation sequence, one molecule of acetyl-CoA, two pairs of electrons, and four H+ ions are removed from the long-chain fatty acyl-CoA, to shorten it by two carbon atoms. The equation for one pass, beginning with the coenzyme A ester of our example, palmitate, is Palmitoyl-CoA + CoA + FAD + NAD+ + H2O myristoyl-CoA + acetyl-CoA + FADH2 + NADH + H+       ............ (16-2) Following removal of one acetyl-CoA unit from palmitoyl-CoA, the coenzyme A thioester of the shortened fatty acid remains, in this case the 14-carbon myristate. The myristoyl-CoA can now enter the β-oxidation sequence and go through another set of four reactions, exactly analogous to the first, to yield a second molecule of acetyl-CoA and lauroylCoA, the coenzyme A thioester of the 12-carbon laurate. Altogether, seven passes through the β-oxidation sequence are required to oxidize one molecule of palmitoyl-CoA to eight molecules of acetyl-CoA (Fig. 16-8b). The overall equation is Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O 8 acetyl-CoA + 7FADH2 + 7NADH + 7H+        ............(16-3) Each molecule of FADH2 formed during oxidation of the fatty acid donates a pair of electrons to ETFP of the respiratory chain (Fig. 16-9); two molecules of ATP are generated during the ensuing transfer of the electron pair to O2 and the coupled oxidative phosphorylations. Similarly, each molecule of NADH formed delivers a pair of electrons to the mitochondrial NADH dehydrogenase; the subsequent transfer of each pair of electrons to O2 results in formation of three molecules of ATP. Thus five molecules of ATP are formed for each two-carbon unit removed in one pass through the sequence as it occurs in animal tissues, such as the liver or heart. Note that water is also produced in this process. Condensation of ADP and Pi releases one H2O for each ATP formed, and transfer of electrons from NADH or FADH2 to O2 yields one H2O per electron pair. R,eduction of O2 by NADH also consumes one H+ per NADH: NADH + H+ + 2O2 NAD+ + H2O. In hibernating animals, fatty acid oxidation provides metabolic energy, heat, and water-all essential for survival of an animal that neither eats nor drinks for long periods (Box 16-1). The overall equation for the oxidation of palmitoyl-CoA to eight molecules of acetyl-CoA, including the electron transfers and oxidative phosphorylation, is Palmitoyl-CoA + 7CoA + 7O2 + 35Pi + 35ADP 8 acetyl-CoA + 35ATP + 42H2O       ............ (16-4) Acetyl-CoA Can Be Further Oxidized via the Citric Acid Cycle The acetyl-CoA produced from the oxidation of fatty acids can be oxidized to CO2 and H2O by the citric acid cycle. The following equation represents the balance sheet for the second stage in the oxidation of our example, palmitoyl-CoA, together with the coupled phosphorylations of the third stage: 8 Acetyl-CoA + 16O2 + 96Pi + 96ADP 8CoA + 96ATP + 104H2O + 16CO2       ............ (16-5) Combining Equations 16-4 and 16-5, we obtain the overall equation for the complete oxidation of palmitoyl-CoA to carbon dioxide and water: Palmitoyl-CoA + 23O2 + 131Pi + 13lADP CoA + 13lATP + l6CO2 + 146H2O       ............ (16-6) Because the activation of palmitate to palmitoyl-CoA consumes two ATP equivalents (p. 484), the net gain per molecule of palmitate is 129 ATP. Table 16-1 summarizes the yields of NADH, FADH2, and ATP in the successive steps of fatty acid oxidation. The standard free-energy change for the oxidation of palmitate to CO2 + H2O is about 9,800 kJ/ mol. Under standard conditions, 30.5 × 129 = 3,940 kJ/mol (about 40% of the theoretical maximum) is recovered as the phosphate bond energy of ATP. However, when the free-energy changes are calculated from actual concentrations of reactants and products under intracellular conditions (see Box 13-2), the free-energy recovery is over 80%; the energy conservation is remarkably efficient.