Fatty acyl carnitine transferase

Figure 7.11 Mechanism of transport of long-chain fatty adds across the inner mitochondrial membrane as fatty acyl carnitine. CRT is the abbreviation for carnitine palmitoyl transferase. CPT-I resides on the outer surface of the inner membrane, whereas CPT-II resides on the inner side of the inner membrane of the mitochondria. Transport across the inner membrane is achieved by a carrier protein known as a translocase. FACN - fatty acyl carnitine, CN - carnitine. Despite the name, CRT reacts with long-chain fatty acids other than palmitate. CN is transported out of the mitochondria by the same translocase.

Answer The transport of fatty acid molecules into mitochondria requires a shuttle system involving a fatty acyl-carnitine intermediate. Fatty acids are first converted to fatty acyl-CoA molecules in the cytosol (by the action of acyl-CoA synthetases) then, at the outer mitochondrial membrane, the fatty acyl group is transferred to carnitine (by the action of carnitine acyl-transferase I). After transport of fatty acyl-carnitine through the inner membrane, the fatty acyl group is transferred to mitochondrial CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no labeled CoA from the cytosolic pool enters the mitochondrion. 

But a transferase now comes upon the scene Making fatty acyl carnitine. 

When large amounts of exogenous fatty acid enter the liver, how can they be transferred into the mitochondrion, for beta oxidation, with malonyl CoA inhibition This dichotomy is overcome because fatty-acyl CoAs inhibit acetyl CoA carboxylase and the malonyl CoA present proceeds onto fatty acids. When the malonyl CoA is converted to fatty acid, the level of malonyl CoA drops and is not restored. Thus, the inhibition of acyl carnitine transferase 1 is removed and fatty-acid oxidation can proceed. 

Fig. 23.5. Transport of long-chain fatty acids into mitochondria. The fatty acyl CoA crosses the outer mitochondrial membrane. Carnitine pahnitoyl transferase I in the outer mitochondrial membrane transfers the fatty acyl group to carnitine and releases Co ASH. The fatty acyl carnitine is translocated into the mitochondrial matrix as carnitine moves out. Carnitine palmitoyl transferase II on the inner mitochondrial membrane transfers the fatty acyl group back to CoASH, to form fatty acyl CoA in the matrix.

Carnitine serves as a cofactor for several enzymes, including carnitine translo-case and acyl carnitine transferases I and II, which are essential for the movement of activated long-chain fatty acids from the cytoplasm into the mitochondria (Figure 11.2). The translocation of fatty acids (FAs) is critical for the genaation of adenosine triphosphate (ATP) within skeletal muscle, via 3-oxidation. These activated FAs become esterified to acylcamitines with carnitine via camitine-acyl-transferase I (CAT I) in the outer mitochondrial membrane. Acylcamitines can easily permeate the membrane of the mitochondria and are translocated across the membrane by carnitine translocase. Carnitine s actions are not yet complete because the mitochondrion has two membranes to cross thus, through the action of CAT II, the acylcar-nitines are converted back to acyl-CoA and carnitine. Acyl-CoA can be used to generate ATP via 3-oxidation, Krebs cycle, and the electron transport chain. Carnitine is recycled to the cytoplasm for fumre use. 

At this point, the acyl-CoA is still in the cytosol of the muscle cell. Entry of the acyl-CoA into the mitochondrial matrix requires two translocase enzymes, carnitine acyl transferase I and carnitine acyl transferase II (CAT I and CAT II), and a carrier molecule called carnitine the carnitine shuttles between the two membranes. The process of transporting fatty acyl-CoA into mitochondria is shown in Figure 7.15. 

Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidized in the mitochondrial matrix. A special transport mechanism is needed to carry long-chain acyl CoA molecules across the inner mitochondrial membrane. Activated long-chain fatty acids are transported across the membrane by conjugating them to carnitine, a zwitterionic alcohol. The acyl group is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyltransferase I (also called carnitine palmitoyl transferase I), which is bound to the outer mitochondrial membrane. 

Carnitine is used mainly for facilitating the transport of long-chain fatty adds into the mitochondria. As shown in Figure4.53, this transport system requires the participation of two different carnitine acyl transferases. One is located on the outside of the mitochondrial membrane, the other on the inner side. Once fatty acyl-camitine is inside the organelle, its carnitine is released. A separate transport system is used to transport this carnitine from the interior of the mitochondrion back to the cytoplasm for reuse. 

The study described in Figure 4.54 illustrates the role of cytosolic malonyl-CoA in controlling the entry of fatty acids into the mitochondrion. The activity of carnitine acyltransferasc w as measured after adding various concentrations of fatty acyl-CoA (0-250 jiM) to a suspension of mitochondria both w ith and without malonyl-CoA. Transferase activity increased with increasing concentrations of fatty acyl-CoA until a plateau was reached. At this point, the transferase was saturated and unable to operate at a faster rate when presented w ith higher concentrations of its substrate. The activity of the transferase w as impaired in the presence of malonyl-CoA. This indicates that an increase in the rate of fatty acid synthesis, with the resultant increase in concentration of malonyl oA, will impair the oxidation of fatty acids. Therefore, the pathways of fatty acid oxidation and synthesis are coordinated. Figure 4-54 also indicates that malonyl-CoA inhibition can be overcome by higher levels of fatty acy)-CoA. Other studies have revealed... 

Fatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneously active. Acetyl CoA carboxylase, the essential control site, is phosphorylated and inactivated by AMP-dependent kinase. The phosphorylation is reversed by a protein phosphatase. Citrate, which signals an abundance of building blocks and energy, partly reverses the inhibition by phosphorylation. Carboxylase activity is stimulated by insulin and inhibited by glucagon and epinephrine. In times of plenty, fatty acyl CoAs do not enter the mitochondrial matrix, because malonyl CoA inhibits carnitine acyl transferase I. 

The answer is b. (Murray, pp 505-626. Scriver, pp 4029-4240. Sack, pp 121-138. Wilson, pp 287-320.) A deficiency in carnitine, carnitine acyl-transferase 1, carnitine acyltransferase 11, or acylcarnitine translocase can lead to an inability to oxidize long-chain fatty acids. This occurs because all of these components are needed to translocate activated long-chain (>10 carbons long) fatty acyl CoA across mitochondrial inner membrane into the matrix where P oxidation takes place. Once long-chain fatty acids are coupled to the sulfur atom of CoA on the outer mitochondrial membrane, they can be transferred to carnitine by the enzyme carnitine acyltransferase I, which is located on the cytosolic side of the inner mitochondrial membrane. Acyl carnitine is transferred across the inner membrane to the matrix surface by translocase. At this point the acyl group is reattached to a CoA sulfhydryl by the carnitine acyltransferase 11 located on the matrix face of the inner mitochondrial membrane. 

The control of fatty-acid oxidation is related to the availability of circulating fatty acids and the activity of palmitoyl carnitine transferase 1. When circulating fatty acids are elevated, considerable fatty-acyl CoA is formed in a number of tissues, including the liver, which is sufficient to inhibit both acetyl CoA carboxylase in the cytosol and, indirectly, pyruvate dehydrogenase in the mitochondrion. Under this condition, neither malonyl CoA nor citrate would accumulate thus, there would be a diminution of fatty-acid synthesis. When large amounts of fatty... 

Carnitine serves as the carrier that transports activated long chain fatty acyl groups across the inner mitochondrial membrane (Fig. 23.4). Carnitine acyl transferases are able to reversibly transfer an activated fatty acyl group from CoA to the hydroxyl group of carnitine to form an acylcamitine ester. The reaction is reversible, so that the fatty acyl CoA derivative can be regenerated from the carnitine ester. 

Carnitine palmitoyltransferase I (CPTI also called carnitine acyltransferase I, CATI), the enzyme that transfers long-chain fatty acyl groups from CoA to carnitine, is located on the outer mitochondrial membrane (Fig. 23.5). Fatty acylcamitine crosses the inner mitochondrial membrane with the aid of a translocase. The fatty acyl group is transferred back to CoA by a second enzyme, carnitine palmitoyl-transferase II (CPTII or CATII). The carnitine released in this reaction returns to the cytosolic side of the mitochondrial membrane by the same translocase that brings fatty acylcamitine to the matrix side. Long-chain fatty acyl CoA, now located within the mitochondrial matrix, is a substrate for (3-oxidation. 

Fatty acid uptake by muscle requires the participation of fatty acid-binding proteins and the usual enzymes of fatty acid oxidation. Fatty acyl-CoA uptake into the mitochondria is controlled by malonyl-CoA, which is produced by an isozyme of acetyl-coA carboxylase (ACC-2 the ACC-1 isozyme is found in liver and adipose tissue and is used for fatty acid biosynthesis). ACC-2 is inhibited by phosphorylation by the AMP-activated protein kinase (AMP-PK) such that when energy levels are low the levels of malonyl CoA will drop, allowing fatty acid oxidation by the mitochondria. In addition, muscle cells also contain the enzyme malonyl CoA decarboxylase, which is activated by phosphorylation by the AMP-PK. Malonyl CoA decarboxylase converts malonyl CoA to acetyl CoA, thereby relieving the inhibition of carnitine palmitoyl transferase I (CPT-I) and stimulating fatty acid oxidation (Fig. 47.5). Muscle cells do not synthesize fatty acids the presence of acetyl CoA carboxylase in muscle is exclusively for regulatory purposes. 

Fig. 47.5. Regulation of fatly acyl CoA entry into muscle mitochondria. 1. Acetyl CoA carboxylase-2 (ACC-2) converts acetyl CoA to malonyl CoA, which inhibits carnitine pahnitoyl transferase I (CPT-I), thereby blocking fatty acyl CoA entry into the mitochondria. 2. However, as energy levels drop, AMP levels rise because of the activity of the adenylate kinase reaction. 3. The increase in AMP levels activates the AMP-activated protein kinase (AMP-PK), which phosphorylates and inactivates ACC-2, and also phosphorylates and activates malonyl CoA decarboxylase (MCoADC). The decarboxylase converts malonyl CoA to acetyl CoA, thereby relieving the inhibition of CPT-1, and allowing fatty acyl CoA entry into the mitochondria. This allows the muscle to generate ATP via the oxidation of fatty acids.

In these experiments cultured glioma cells were exposed to TTA, and key enzymes in peroxisomal (fatty acyl-CoA oxidase—FAO) and mitochondrial (carnitine palmitoyl transferase II—CPT II) fatty acid oxidation were studied. 

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. 

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... 

As well as being the substrate for fatty acid synthesis, malonyl CoA has an important role in controlling (3-oxidation of fatty acids. Malonyl CoA is a potent inhibitor of carnitine palmitoyl transferase 1, the mitochondrial outer membrane enzyme that regulates uptake of fatty acyl CoA into the mitochondria (section 5.5.1). This means that, under conditions in which fatty acids are being synthesized in the cytosol, there will not be uptake into the mitochondria for (3-oxidation. (See also section 10.6.2.1 for a discussion of the role of malonyl CoA in regulating muscle fuel selection.)... 

P-Oxidation of fatty acids is controlled by the uptake of fatty acids into the mitochondria - as discussed in section 5.5.1, this is controlled by the activity of carnitine acyl transferase on the outer mitochondrial membrane, and by the countertransport of acyl-carnitine and free carnitine across the inner mitochondrial membrane. 

In recent years the inner mitochondrial membrane has been commonly recognized as the barrier separating the locus of fatty acyl-CoA oxidation from other biochemical events of the cell. This barrier appears to be impermeable to CoASH and to CoASH derivatives. As a result the fatty acyl-CoA externally added to intact mitochondria are not oxidized, unless carnitine is present in the system. The existence of carnitine acyl transferase ... 

CoASH and thus reforming the true immediate substrate for the oxidatioa According to this hypothesis, acyl carnitine, which is not a substrate for the enzymes of the oxidative process, is an admirable substrate for fatty acid degradation in intact mitochondrial systems. In addition, mitochondria depleted of their endogenous energy donors (ATP, GTP) are unable to oxidize add fatty acids unless ATP and carnitine are both present in the system. This observation clearly proves that acyl-CoA is formed outside of the inner mitochondrial membrane—i.e. outside of the oxidation compartment—and is transported to the inner compartment via the carnitine-linked transport mechanism. In agreement with these results an ATP-dependent thiokinase has been identified in the outer mitochondrial membrane, and an acyl-camitine transferase has been found in the inner mitochondrial membrane. 

Acyl-CoA synthetase activity towards long-chain fatty-acid substrates is present in the outer mitochondrial membrane. However, fatty acyl-CoAs do not readily traverse biological membranes such as the inner mitochondrial membrane. A highly sophisticated transport system has evolved to allow tight regulation of fatty-acid entry into the mitochondrion (Figure 2). Carnitine palmitoyl transferase 1 (CPTl), located on the inner aspect of the... 

The steps in the subsequent utilization of muscle LCFAs may be summarized as follows. The free fatty acids, liberated from triglycerides by a neutral triglyceride lipase, are activated to form acyl CoAs by the mediation of LCFAcyl-CoA synthetase which is situated on the outer mitochondrial membrane. The next step involves carnitine palmitoyl transferase I (CPT I, see Figure 9) which is also located on the outer mitochondrial membrane and catalyzes the transfer of LCFAcyl residues from CoA to carnitine (y-trimethyl-amino-P-hydroxybutyrate). LCFAcyl... 

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