Digestion, Mobilization, and Transport of Fats:- Fatty Acids Are Activated and Transported into Mitochondria
The enzymes of fatty acid oxidation in animal cells are located in the mitochondrial matrix, as demonstrated in 1948 by Eugene P. Kennedy and Albert Lehninger. The fatty acids with chain lengths of 12 or fewer carbons enter mitochondria without the help of membrane trans porters. Those with 14 or more carbons, which constitute the majority of the FFA obtained in the diet or released from adipose tissue, cannot pass directly through the mitochondrial membranes—they must first undergo the three enzymatic reactions of the carnitine shuttle. The first reaction is catalyzed by a family of isozymes (different isozymes specific for fatty acids having short, intermediate, or long carbon chains) present in the outer mitochondrial membrane, the acyl-CoA synthetases, which promote the general reaction
Fatty acid+ CoA+ ATP⇌ fatty acyl–CoA+ AMP+ PPi
Thus, acyl-CoA synthetases 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, coupled to the cleavage of ATP to AMP and PPi. (Recall the description of this reaction in Chapter 13, to illustrate how the free energy released by cleavage of phosphoanhydride bonds in ATP could be coupled to the formation of a high-energy compound; p. XXX.) The reaction occurs in two steps and involves a fatty acyl–adenylate intermediate (Fig. 17–5). Fatty acyl–CoAs, like acetyl-CoA, are high-energy compounds; their hydrolysis to FFA and CoA has a large, negative standard free-energy change (ΔG0 ≈ 31 kJ/mol). The formation of a fatty acyl–CoA is made more favorable by the hydrolysis of two high-energy bonds in ATP; the pyrophosphate formed in the activation reaction is immediately hydrolyzed by inorganic pyrophosphatase (left side of Fig. 17–5), which pulls the preceding activation reaction in the direction of fatty acyl–CoA formation. The overall reaction is
Fatty acid+ CoA +ATP→ fatty acyl–CoA+AMP+2Pi (17–1) ΔG0=-34 kJ/mol
Fatty acyl–CoA esters formed at the cytosolic side of the outer mitochondrial membrane can be trans ported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize
MECHANISM FIGURE 17–5 Conversion of a fatty acid to a fatty acyl–CoA. The conversion is catalyzed by fatty acyl–CoA synthetase and inorganic pyrophosphatase. Fatty acid activation by formation of the fatty acyl–CoA derivative occurs in two steps. In step 1, the carboxylate ion displaces the outer two (β and β) phosphates of ATP to form a fatty acyl–adenylate, the mixed anhydride of a carboxylic acid and a phosphoric acid. The other product is PPi, an excellent leaving group that is immediately hydrolyzed to two Pi, pulling the reaction in the forward direction. In step 2, the thiol group of coenzyme A carries out nucleophilic attack on the enzyme-bound mixed anhydride, displacing AMP and forming the thioester fatty acyl–CoA. The overall reaction is highly exergonic.
FIGURE 17–6 Fatty acid entry into mitochondria via the acyl-carnitine/ carnitine transporter. After fatty acyl–carnitine is formed at the outer membrane or in the intermembrane space, it moves into the matrix by facilitated diffusion through the transporter in the inner membrane. In the matrix, the acyl group is transferred to mitochondrial coenzyme A, freeing carnitine to return to the intermembrane space through the same transporter. Acyltransferase I is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis (see Fig. 21–1). This inhibition prevents the simultaneous synthesis and degradation of fatty acids.
membrane lipids. Fatty acids destined for mitochondrial oxidation are transiently attached to the hydroxyl group of carnitine to form fatty acyl–carnitine—the second reaction of the shuttle. This transesterification is cat alyzed by carnitine acyltransferase I(Mr88,000), in the outer membrane. Either the acyl-CoA passes through the outer membrane and is converted to the carnitine ester in the intermembrane space (Fig. 17–6), or the carnitine ester is formed on the cytosolic face of the outer membrane, then moved across the outer mem brane to the intermembrane space—the current evidence does not reveal which. In either case, passage into the intermembrane space (the space between the outer and inner membranes) occurs through large pores (formed by the protein porin) in the outer membrane. The fatty acyl–carnitine ester then enters the matrix by facilitated diffusion through the acyl-carnitine/carnitine transporter of the inner mitochondrial membrane (Fig. 17–6).
In the third and final step of the carnitine shuttle, the fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II. This isozyme, located on the inner face of the inner mitochondrial membrane, re generates fatty acyl–CoA and releases it, along with free carnitine, into the matrix (Fig. 17–6). Carnitine reenters the intermembrane space via the acyl-carnitine/carnitine transporter.
This three-step process for transferring fatty acids into the mitochondrion—esterification to CoA, transes terification to carnitine followed by transport, and trans esterification back to CoA—links two separate pools of coenzyme A and of fatty acyl–CoA, one in the cytosol, the other in mitochondria. These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids (see Fig. 21–10). Fatty acyl–CoA in the cytosolic pool can be used for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP pro duction. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. The carnitine-mediated entry process is the rate limiting step for oxidation of fatty acids in mitochondria and, as discussed later, is a regulation point. Once inside the mitochondrion, the fatty acyl–CoA is acted upon by a set of enzymes in the matrix.
