Carnitine enzymes

Ascorbic acid is involved in carnitine biosynthesis. Carnitine (y-amino-P-hydroxybutyric acid, trimethylbetaine) (30) is a component of heart muscle, skeletal tissue, Uver and other tissues. It is involved in the transport of fatty acids into mitochondria, where they are oxidized to provide energy for the ceU and animal. It is synthesized in animals from lysine and methionine by two hydroxylases, both containing ferrous iron and L-ascorbic acid. Ascorbic acid donates electrons to the enzymes involved in the metabohsm of L-tyrosine, cholesterol, and histamine (128). 

All of the other enzymes of the /3-oxidation pathway are located in the mitochondrial matrix. Short-chain fatty acids, as already mentioned, are transported into the matrix as free acids and form the acyl-CoA derivatives there. However, long-chain fatty acyl-CoA derivatives cannot be transported into the matrix directly. These long-chain derivatives must first be converted to acylearnitine derivatives, as shown in Figure 24.9. Carnitine acyltransferase I, located on the outer side of the inner mitochondrial membrane, catalyzes the formation of... 

Figure 3. Mitochondrial fatty acid oxidation. Long-chain fatty acids are converted to their CoA-esters as described in the text, and their fatty-acyl-groups transferred to CoA in the matrix by the concerted action of CPT 1, the acylcarnitine/carnitine exchange carrier and CPT (A) as described in the text. Medium-chain and short-chain fatty acids (Cg or less) diffuse directly into the matrix where they are converted to their acyl-CoA esters by a acyl-CoA synthase. The mechanism of p-oxidation is shown below (B). Each cycle of P-oxidation removes -CH2-CH2- as an acetyl unit until the fatty acids are completely converted to acetyl-CoA. The enzymes catalyzing each stage of P-oxidation have different but overlapping specificities. In muscle mitochondria, most acetyl-CoA is oxidized to CO2 and H2O by the citrate cycle (Figure 4) some is converted to acylcamitine by carnitine acetyltransferase (associated with the inner face of the inner membrane) and exported from the matrix. Some acetyl-CoA (if in excess) is hydrolyzed to acetate and CoASH by acetyl-CoA hydrolase in the matrix. Enzymes ...

Increased fatty acid oxidation is a characteristic of starvation and of diabetes meUims, leading to ketone body production by the Ever (ketosis). Ketone bodies are acidic and when produced in excess over long periods, as in diabetes, cause ketoacidosis, which is ultimately fatal. Because gluconeogenesis is dependent upon fatty acid oxidation, any impairment in fatty acid oxidation leads to hypoglycemia. This occurs in various states of carnitine deficiency or deficiency of essential enzymes in fatty acid oxidation, eg, carnitine palmitoyltransferase, or inhibition of fatty acid oxidation by poisons, eg, hypoglycin. 

Carnitine (p-hydroxy-y-trimethylammonium butyrate), (CHjljN"—CH2—CH(OH)—CH2—COO , is widely distributed and is particularly abundant in muscle. Long-chain acyl-CoA (or FFA) will not penetrate the inner membrane of mitochondria. However, carnitine palmitoyltransferase-I, present in the outer mitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner membrane and gain access to the P-oxidation system of enzymes (Figure 22-1). Carnitine-acylcar-nitine translocase acts as an inner membrane exchange transporter. Acylcarnitine is transported in, coupled with the transport out of one molecule of carnitine. The acylcarnitine then reacts with CoA, cat-... 

A number of iron-containing, ascorbate-requiring hydroxylases share a common reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of a-ketoglutarate (Figure 28-11). Many of these enzymes are involved in the modification of precursor proteins. Proline and lysine hydroxylases are required for the postsynthetic modification of procollagen to collagen, and prohne hydroxylase is also required in formation of osteocalcin and the Clq component of complement. Aspartate P-hydroxylase is required for the postsynthetic modification of the precursor of protein C, the vitamin K-dependent protease which hydrolyzes activated factor V in the blood clotting cascade. TrimethyUysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine. 

Inborn errors of fatty acid oxidation Carnitine entry into cells and mitochondria Certain enzymes of fatty acid oxidation... 

The transport is accomplished with the participation of carnitine, which takes up the acyl from acyl-CoA on the outer membrane side. Acylcamitine assisted by carnitine translocase diffuses to the inner side of the membrane to give its acyl to the CoA located in the matrix. The process of reversible acyl transfer between CoA and carnitine on the outer and inner sides of the membrane is effected by the enzyme acyl-CoA-camitine transferase. 

Production of Malonyl-CoA for the Fatty Acid Biosynthesis. Acetyl-CoA serves as a substrate in the production of malonyl-CoA. There are several routes by which acetyl-CoA is supplied to die cytoplasm. One route is the transfer of acetyl residues from the mitochondrial matrix across the mitochondrial membrane into the cyto-plasm. This process resembles a fatty acid transport and is likewise effected with the participation of carnitine and the enzyme acetyl-CoA-camitine transferase. Another route is the production of acetyl-CoA from citrate. Citrate is delivered from the mitochondria and undergoes cleavage in the cytoplasm by the action of the enzyme ATP-citrate lyase ... 

Carnitine palmitoyltransferase deficiency is an autosomal recessive myopathy caused by a genetic defect of the mitochondrial enzyme CPT (Fig. 42-2). The disease is prevalent in men (male female ratio, 5.5 1) and appears to be the most common cause of recurrent myoglobinuria in adults [4]. 

Two studies were located in which rats received di- -octylphthalate dietary exposures of 250 or 500 mg/kg/day for either 10 or 26 weeks (Carter et al. 1992 DeAngelo et al. 1986). Five male rats were first initiated with a single subcarcinogenic intraperitoneal dose of diethylnitrosamine (30 mg/kg), followed by partial hepatectomy. Di-w-octylphthalatc caused substantial increases in gamma-glutamyltranspeptidase (GGT) positive liver foci when compared with the controls (e.g., from 3.5 to 20.8 foci/cm2) or in hepatic levels of marker enzymes for altered cellular foci (GGT and glutathione 5-transferase [GST]). Only a slight increase (threefold) was observed for carnitine acetyltransferase (CAT) activity, a marker for peroxisome... 

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. 

The physiological functions of carboxylesterases are still partly obscure but these enzymes are probably essential, since their genetic codes have been preserved throughout evolution [84] [96], There is some evidence that microsomal carboxylesterases play an important role in lipid metabolism in the endoplasmic reticulum. Indeed, they are able to hydrolyze acylcamitines, pal-mitoyl-CoA, and mono- and diacylglycerols [74a] [77] [97]. It has been speculated that these hydrolytic activities may facilitate the transfer of fatty acids across the endoplasmic reticulum and/or prevent the accumulation of mem-branolytic natural detergents such as carnitine esters and lysophospholipids. Plasma esterases are possibly also involved in fat absorption. In the rat, an increase in dietary fats was associated with a pronounced increase in the activity of ESI. In the mouse, the infusion of lipids into the duodenum decreased ESI levels in both lymph and serum, whereas an increase in ES2 levels was observed. In the lymph, the levels of ES2 paralleled triglyceride concentrations [92] [98],... 

Enzymatic hydroxylation of biological molecules is often catalyzed by hydroxylases. These types of enzymes are either oxygenases or peroxidases, in which the source of oxygen is O2 or H2O2, respectively. Cytochrome P-450-dependent enzymes represent a common class of enzymes that carry out hydroxylation reactions. L-Carnitine is a metabolite isolated from many organisms and its biosynthesis begins with the enzymatic hydroxylation of trimethyllysine. The intermediate, 3-hydroxyl-e-(A(A(ALtrimethyl)-L-lysine, is further... 

Mitochondria contain all the enzymes necessary for oxidation of fatty acids but, before this can take place, the fatty acids have to be transported into the mitochondria. Transport requires the formation of an ester of the fatty acid with a compound, carnitine, in a reaction catalysed by the enzyme carnitine palmitoyltransferase ... 

It is the fatty acyl-camitine that is transported across the inner mitochondrial membrane from the cytosol to the matrix so that two different enzymes are reqnired for the transport. The first enzyme, carnitine palmitoyltransferase-1, is located on the outer surface of this membrane and the second enzyme, carnitine pahnitoyltransferase-II, is located on the inner side of this membrane (Figure 7.11). Carnitine may have this role since it is smaller than CoASH and has no net charge. 

The activity of carnitine palmitoyltransferase-I plays an important role in the regulation of fatty acid oxidation malonyl-CoA is an allosteric exhibitor of the enzyme. Malonyl-CoA is a key intermediate in fatty acid synthesis, which ensures that fatty acid oxidation is decreased when synthesis is taking place. Nonetheless, malonyl-CoA has a major role in the control of fatty acid oxidation in all tissues in which fatty acid oxidation occurs, even if no synthesis takes place. 

Figure 7.15 Inhibition of acetyl-CoA carboxylase by cyclic AMP dependent protein kinase and AMP dependent protein kinase the dual effect of glucagon. Phosphorylation of acetyl-CoA carboxylase by either or both enzymes inactivates the enzyme which leads to a decrease in concentration of malonyl-CoA, and hence an increase in activity of carnitine palmitoyltransferase-I and hence an increase in fatty acid oxidation. Insulin decreases the cyclic AMP concentration maintaining an active carboxylase and a high level of malonyl-CoA to inhibit fatty acid oxidation.

There is a marked increase in the activity of the key enzymes that convert fatty acids into ketone bodies carnitine palmitoyltransferase and HMG-CoA synthase. 

ATP. The presence of acetyl-carnitine in mitochondria, at a concentration 400-fold greater than that of acetyl-CoA, along with the enzyme carnitine acetyltransferase, acts as a buffer to protect against large changes in the concentration of acetyl-CoA ... 

The inner mitochondrial membrane has a group-specific transport system for fatty acids. In the cytoplasm, the acyl groups of activated fatty acids are transferred to carnitine by carnitine acyltransferase [1 ]. They are then channeled into the matrix by an acylcar-nitine/carnitine antiport as acyl carnitine, in exchange for free carnitine. In the matrix, the mitochondrial enzyme carnitine acyltransferase catalyzes the return transfer of the acyl residue to CoA. 

This enzyme [EC 2.3.1.7], also known as carnitine acet-ylase, catalyzes the reversible reaction of acetyl-CoA with carnitine to yield coenzyme A and O-acetylcarni-tine. The enzyme can also use propanoyl-CoA and buta-noyl-CoA as substrates. 

C. Long-chain fatty acids (LCFAs), which have carbon chain lengths of 12-22 units (C12-C22), must be transported into the mitochondrial matrix where the enzymes responsible for their oxidation are located. This is accomplished by the carnitine shutde (Figure 8-3). 

In 1955, Fritz determined that carnitine plays an essential role in fatty acid -oxidation (FAO), and in 1973 the first two clinically relevant disorders affecting this pathway were described primary carnitine deficiency by Engel and Angelini, and carnitine palmitoyltransferase (CPT) type II (CPT-II) deficiency by DiMauro and DiMauro [6, 7]. To date, more than 20 different enzyme deficiency states affecting fatty acid transport and mitochondrial / -oxidaLion have been described [8] and additional enzymes involved in this pathway are still being discovered [9, 10]. 

Cultured fibroblasts or amniocytes can be probed with FAO substrates and carnitine. Cell cultures deficient of an FAO enzyme will accumulate specific acylcarni-tine species when incubated with substrates such as palmitate, allowing for the diagnosis of FAO disorders [28-37]. Modifications of this assay system have also been developed for the diagnosis of defects affecting the metabolism of branched-chain amino acids [20, 31, 34]. Recently, this approach was also adapted for the study of peripheral blood mononuclear cells [38]. 

Carnitine and its esters are present in all biological fluids, and depending on the enzyme defect, a particular acylcarnitine pattern becomes apparent where those acyl-carnitine species serving as direct substrates for the defective enzyme accumulate disproportionately to the down- and upstream metabolites (Table 3.2.1). 

With the clofibrate type of inducer, other changes are also apparent. Thus, there is a proliferation in the number of peroxisomes (an intracellular organelle) as well as induction of a particular form of cytochrome P-450 involved in fatty acid metabolism. A number of other enzymes associated with the role of this organelle in fatty acid metabolism are also increased, such as carnitine acyltransferase and catalase. This phenomenon is discussed in more detail in chapter 6. 

Figure 7.15 The interaction between valproate and the mitochondrial p-oxidation system. Dark arrows denote depletion. Filled in circle is the carnitine transporter. 2,4, VPA 2,4, diene-VPA CoA ester. This reactive metabolite damages the enzyme enoyl CoA hydratase and the mitochondrial membranes and depletes GSH, as indicated.

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 transporters. 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... 

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