P-oxidation spiral

Each turn of the P-oxidation spiral splits off a molecule of acetyl-CoA. The process involves four enzymes catalysing, in turn, an oxidation (to form a double bond), a hydration, another oxidation (forming a ketone from a secondary alcohol) and the transfer of an acetyl group to coenzyme A (Figure 7.12). The process of P-oxidation operates as a multienzyme complex in which the intermediates are passed from one enzyme to the next, i.e. there are no free intermediates. The number of molecules of ATP generated from the oxidation of one molecule of the long-chain fatty acid pal-mitate (C18) is given in Table 7.4. Unsaturated fatty acids are also oxidised by the P-oxidation process but require modification before they enter the process (Appendix 7.3). 

E) Two acetyl CoA molecules are produced in each turn of the p-oxidation spiral... 

In the first of four reactions that constimte one cycle of the P-oxidation spiral, acyl-CoA is dehydrogenated to 2-tra/w-enoyl-CoA according to the following equation. 

The oxidation of fatty acids to acetyl CoA in the p-oxidation spiral conserves energy as FAD(2H) and NADH. FAD(2H) and NADH are oxidized in the electron transport chain, generating ATP from oxidative phosphorylation. Acetyl CoA is oxidized in the TCA cycle or converted to ketone bodies. 

The p-oxidation spiral uses the same reaction types seen in the TCA cycle when succinate is converted to oxaloacetate. 

P-carbon during the p-oxidation spiral, and can no longer interfere with oxidation of the p-carbon. Peroxisomal p-oxidation thus can proceed normally, releasing pro-pionyl CoA and acetyl CoA with alternate turns of the spiral. When a medium chain length of approximately eight carbons is reached, the fatty acid is transferred to the mitochondrion as a carnitine derivative, and p-oxidation is resumed. 

One property of the translocase is specially noteworthy its affinity for long-chain acylcamitine is very much higher than for carnitine itself. This suggests a basic role for carnitine, additional to the acetylation buffer already mentioned. Every fourth reaction of the P-oxidation spiral (the excision of an acetyl unit by a thiolase enz5mie) requires free CoASH, so that if the CoA of the mitochondrial matrix became over-acy-lated the whole process would come to a halt. If acyl-CoA were formed directly from fatty acids in the same compartment (the matrix) as oxidation this might readily happen. However, the carnitine system guards against this fail-nonsafe situation, because if the acyl-CoA/CoASH ratio (and hence the acylcamitine/camitine ratio) rises the translocase will selectively export acylcamitine from the matrix and import carnitine this will lower the ratio and restore P-oxidation. It remains (I think ) to be seen if this effect can be conclusively demonstrated. 

Although the fatty acid p-oxidation spiral comprises only four reactions, imder-standing of the complexity of fatty acid degradation has dramatically increased in recent years due to the discovery of a variety of new P-oxidation enzymes. This article will discuss the enzymes that catalyze the second and third steps of the P-oxidation pathway, an area of recent and substantial progress. 

The second step of the P-oxidation spiral is the reversible hydration of 2-trans-enoyl-CoA to yield L-3-hydroxyacyl-CoA, catalyzed by enoyl-CoA hydratase. However, in fungi 2-tra 5-enoyl-CoA is hydrated by peroxisomal D-3-hydroxyacyl-CoA dehydratase to form D-3-hydroxyacyl-CoA. Enoyl-CoA hydratases are usually associated with the N-terminal region of multifunctional proteins except for the mitochondrial matrix enoyl-CoA hydratase and the E. coli long-chain enoyl-CoA hydratase (see Table 1). D-3-hydioxyacyl-CoA dehydratases are located on the C-terminal domain of the peroxisomal D-specific bifunctional P-oxidation enzyme or the central domain of 1 P-hydroxysteroid dehydrogenase type IV. ... 

The discovery of A -A -enoyl-CoA isomerase gave rise to the proposal that metabolites with odd-numbered double bonds underwent only a >-cis to 2-trans isomerization before re-entering the p-oxidation spiral (Fig. 2). However, an additional pathway that reduces the double bond in an NADPH-dependent manner was recently describe by Tsemg and Jin." The starting metabolite in this reaction sequence is /ra 5-2-c/5-5-dienoyl-CoA, which can either complete the P-oxidation cycle to yield c/5-3-enoyl-CoA or can be converted to /ra 5-3,cw-5-dienoyl-CoA by A AAenoyl-CoA isomerase. A novel enzyme, AJ.5 2.4.dienoyl-CoA isomerases then catalyzes the shift of both double bonds to produce a tran5-2,4- a 5-dienoyl-CoA" " which is a substrate of 2,4-dienoyl-CoA reductase and, hence, can be reduced to />-a .s-3-enoyl-CoA. This is then converted to trans-2-enoy -CoA by A A -enoyl-CoA isomerase (Fig. 2). 

Mitochondrial P-oxidation of long-chain fatty acids is the major source of energy production in man. The mitochondrial inner membrane is impermeable to long chain fatty acids or their CoA esters whereas acylcamitines are transported. Three different gene products are involved in this carnitine dependent transport shuttle carnitine palmi-toyl transferase I (CPT I), carnitine acyl-camitine carrier (CAC) and carnitine palmitoyl transferase II (CPT II). The first enzyme (CPT I) converts fatty acyl-CoA esters to their carnitine esters which are subsequently translocated across the mitochondrial inner membrane in exchange for free carnitine by the action of the carnitine acyl-camitine carrier (CAC). Once inside the mitochondrion, CPT II reconverts the carnitine ester back to the CoA ester which can then serve as a substrate for the P-oxidation spiral. 

As a stearoyl CoA molecule (18 carbons) passes through the p-oxidation spiral, 9 acetyl CoA, 8 FADH2, and 8 NADH molecules are produced. Acetyl CoA produced in the fatty acid spiral can enter the citric acid cycle (followed by the electron transport chain), where each molecule of acetyl CoA results in the production of 10 ATP molecules. In addition, when the FADH2 and NADH molecnles enter the electron transport chain, each FADH2 yields 1.5 ATP molecules, and each NADH yields 2.5 molecules. The calculations are summarized in I Table 14.1, which shows a total of 120 molecules of ATP formed from the 18-carbon fatty acid. 

Whereas the mitochondrial enzymes of p-oxidation reside within the area bound by inner membrane, activation of fatty acids proceeds largely at sites exterior to this membrane. The transport of activated acyl groups across the inner mitochondrial membrane Is brought about by a carnitine dependent route (Fritz, 1963 Bremer, 1968 Bressler, 1970). A carnitine acyltransferase localized on the outer aspect of inner membrane utilizes cytosolic free carnitine to convert the cytosolic acyl-CoA to cytosolic acylcarnitine (Fig. 1). A translocase of the inner membrane then moves the acylcarnitine inside in exchange for the simultaneous movement of carnitine in the opposite direction. Another carnitine acyltransferase, situated on the inner side of the inner membrane, utilizes matrix CoA to convert acylcarnitine to acyl-CoA, thus producing the latter in the same compartment where enzymes of the p-oxidation spiral exist (Pande, 1975 Ramsay and Tubbs, 1975 Tubbs and... 

In the degradation of normally occurring unsaturated fatty acids with cis double bonds, intermediates that are not on the direct path of P-oxidation are produced, and their channeling into the p-oxidation spiral entails addi-... 

Evidence so far indicates that substrate and cofactor availability, and product inhibition, constitute the main modes for the regulation of fatty acid oxidation within mitochondria there is nothing to indicate that substrate flow over the p-oxidation spiral is further regulated by allosteric or covalent modification of the activity of any enzyme of this spiral. As is to be expected for a multistep metabolic pathway, diverse steps contribute to the overall regulation of fatty acid oxidation in different tissues under different conditions. /S/lore is known about this process in liver and heart than in other tissues and its brief description follows. 

A major determinant of the mitochondrial fatty acid oxidation normally in liver is the delivery rate of activated fatty acyl groups to the enzymes of the P-oxidation spiral in the matrix. Although the importance of the delivery of free fatty acids to liver is well established in this regard, the fact that at times intracellular lipolysis alone can provide enough free fatty acids for oxidation needs to be appreciated. The observations that livers from diabetic rats continue to produce ketone bodies nearly maximally, even when perfused with medium lacking fatty acids, attests to the ability of intracellular lipolysis to furnish substrates for p-oxidation for appreciable periods, at least in liver, and to its activation in the diabetic state (Krebs et aL, 1969 Van Harken et aL, 1969) indirect evidences indicate that a cyclic AMP dependent hormone sensitive lipase exists in liver (for references, see Lund et aL, 1980). 

Fatty acid oxidation occurs in mitochondria, and therefore the fatty acid substrate (in the form of fatty acyl CoA) needs to be transported across the mitochondrial membranes. Short- and medium-chain fatty acids can readily penetrate mitochondria. Long-chain acyl-CoA are able to cross the mitochondrial outer membrane, but cannot penetrate the inner membrane. Translocation of these is a carnitine-dependent process involving the coordinate action of isoforms of carnitine palmitoyl transferase on the mitochondrial outer and inner membranes (see Gurr et aL, 2002). Fatty acid oxidation itself involves the progressive removal of 2-carbon units, as acetyl-CoA, from the carboxyl end of the acyl-CoA (Gurr et aL, 2002). It is often termed the p-oxidation spiral since... 

The sequence of four enzyme reactions shown in the diagram below results in the removal of two pairs of hydrogen atoms from the fatty acyl CoA molecule which are passed to the cofactors NAD+ and FAD which become reduced to NADH and FADH2. As these reactions occur in the mitochondria, it is easy for the cofactors to be rapidly reoxidized by the electron transport process (the cytochrome chain) and this results in ATP synthesis. A molecule of acetyl CoA is produced per turn of the p-oxidation spiral. This acetyl CoA can be further metabolized to CO2, but cannot be used as a source of intermediates for glucose synthesis by gluconeogenesis. 

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