Carboxylation of propionyl CoA

Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase (Figure 45-14) are vitamin Bj2-dependent enzymes. Methylmalonyl CoA is formed as an intermediate in the catabolism of valine and by the carboxylation of propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely, fatty acids with an odd number of carbon atoms—or directly from propionate, a major product of microbial fer-... 

Carboxylation of propionyl-CoA is accomplished by propionyl-CoA carboxylase (biotin, which is the carboxyl group carrier, serves as a coenzyme for this enzyme) the presence of ATP is also required. The methylmalonyl-CoA formed is converted by methylmalonyl-CoA mutase (whose coenzyme, deoxyadenosylcobalamin, is a derivative of vitamin B]2) to succinyl-CoA the latter enters the Krebs cycle. 

Propionate formation in cestodes probably proceeds essentially via reversal of the reactions required for the conversion of propionate to succinate in animal tissues (Fig. 5.10). Two of the enzymes involved, propionyl-CoA carboxylase and methylmalonyl-CoA mutase have been demonstrated in the mitochondria of S. mansonoides (643, 884). An acyl-CoA carboxylase, which can catalyse the carboxylation of propionyl-CoA, has also been isolated from this worm (533). The proposed pathway of propionate formation is associated with net ATP synthesis... 

The steric course of the methylmalonyl-CoA mutase reaction, as it affects the C-2 of methylmalonyl-CoA, can be elucidated by determining the absolute configuration of the substrate and of a suitably labelled product. The former problem was solved by two groups [30,31]. Briefly, the (25) configuration of the epimeric product obtained from enzymic carboxylation of propionyl-CoA was established and the (2R) configuration of the mutase-active methylmalonyl-CoA followed per ex-clusionem. 

Sprinson and coworkers [30] conducted the methylmalonyl-CoA mutase reaction in deuterium oxide using a crude mitochondrial preparation. The presence of methylmalonyl-CoA epimerase insured that (1) all substrate molecules incorporated one atom of deuterium into position 2, and (2) in the course of the reaction the (2R)-epimer of methylmalonyl-CoA was continuously supplied by epimerization of the (25)-epimer, which was in turn generated by the enzymic carboxylation of propionyl-CoA. Alkaline hydrolysis of the product and subsequent purification furnished succinic acid which was mainly monodeuterated (70% 2H,-, 15% 2H2-labelled and 13% unlabelled species). A positive ORD curve revealed its (5) configuration indicating stereochemical retention for the AdoCbl-dependent rearrangement (Fig. 22). No plausible explanation could be offered for the formation of doubly deuterated and unlabelled species. Essentially the same results were later obtained with a highly purified mutase preparation from Propionibacterium sher-manii (J. Retey, unpublished). 

Propionyl CoA Carboxylase Propionyl CoA carboxylase catalyzes the carboxylation of propionyl CoA to methyhnalonyl CoA, which undergoes a vitamin Bi2-dependent isomerization to succinyl CoA (see Figure 10.13). This reaction provides a pathway for the oxidation, through the tricarboxylic acid cycle, of propionyl CoA arising from the catabolism of isoleucine, valine, odd-carbon fatty acids, and the side chain of cholesterol. 

This step is the culmination of a reaction sequence in which propionyl-CoA, a toxic metabolite derived from the degradation of fats, is removed from circulation. Carboxylation of propionyl-CoA gives (5)-methylmalonyl-CoA, which is epimerized to (/ )-methylmalonyl-CoA. Conversion of the (7 )-isomer to succinyl-CoA allows further metabolism via the Krebs cycle [74]. Methylmalonyl-CoA mutase is also the only adenosylcobalamin-dependent enzyme known to participate in human metabolism, and as such has received significant study [30, 37, 38]. 

MCM plays an essential role in propionate metabolism. Propionate and propionyl-CoA are intermediates in the catabolism of leucine and isoleucine and are further metabolized by carboxylation of propionyl-CoA to methylmalonyl-CoA. Isomerization to succinyl-CoA feeds the carbon chain into the tricarboxylic acid pathway of oxidative metabolism. For this reason, MCM is an important enzyme in bacterial and mammalian metabolism. It is one of the two vitamin Bj2-dependent enzymes known to be important in human metabolism. 

Propionyl-CoA carboxylase catalyzes the carboxylation of propionyl-CoA to form D-methylmalonyl-CoA in propionic acid metabolism, as shown in the reaction that follows ... 

Fatty acids containing an odd number of carbon atoms undergo -oxidation, producing acetyl CoA, until the last spiral, when five carbons remain in the fatty acyl CoA. In this case, cleavage by thiolase produces acetyl CoA and a three-carbon fatty acyl CoA, propionyl CoA (Fig. 23.10). Carboxylation of propionyl CoA yields methylmalonyl CoA, which is ultimately converted to succinyl CoA in a vitamin B 12-dependent reaction (Fig. 23.11). Propionyl CoA also arises from the oxidation of branched chain amino acids. 

Provided that the fatty acid contains an even number of C-atoms, it can be totally degraded to acetyl-CoA by p-oxidation. If the fatty acid contains an uneven number of C-atoms, however, the stepwise removal of two carbons at a time by p-oxidation eventually leads to a Cj compound, i.e. propionyl-CoA, which must be metabolized by alternative pathways. These pathways are shown in Rg.2 considerable quantities of propionyl-CoA also arise from the degradation of the branched-chain amino acids, isoleucine and valine (see L-leucine). The main pathway involves carboxylation of propionyl-CoA to methylma-lonyl-CoA, followed by the vitamin B]2-dependent isomerization of methylmalonyl-CoA to sucdnyl-... 

The metabolic pathway for propionic acid use in mammals involves the carboxylation of propionyl CoA in the presence of a specific carboxylase and ATP. The products of the reaction are methylmalonyl CoA and ADP. The ultimate product of propionate metabolism is succinate, which is oxidized via the Krebs cycle. These reactions all occur in the mitochondria. The ability of the mitochondria of biotin-deficient animals to use propionic acid is reduced. This metabolic defect of the deficient mitochondria cannot be corrected by adding biotin in vitro, but the rate of propionic acid use by mitochondria is restored to normal if the deficient animals are fed a normal diet containing biotin. 

Mammalian mitochondria metabolize propionyl-CoA to succinyl-CoA. This pathway involves the carboxylation of propionyl-CoA in a biotin-dependent reaction to yield methylmalonyl-CoA. In a subsequent mutase... 

Tietz and Ochoa could detect no biotin in their enzyme preparation although in biotin deficiency carboxylation of propionyl CoA is greatly diminyied. After it was established that biotin is the coenzyme of the carboxylation enzyme of jS-methylcrotonyl CoA lOJf) it was soon shown that this is also true for the propionyl CoA carboxylase 105a, lOSb). 

PCCase is a biotinylated protein that catalyzes a reaction required in the catabolism of amino acids and fatty acids of odd-numbered chain length, and in the catabolism and anabolism of branched-chain fatty acids. In order to characterize the structure of this enzyme from plants we undertook its purification. PCCase activity was purified from extracts of maize leaves by a four step scheme that included PEG precipitation, hydrophobic interaction chromatography, anion exchange chromatography and affinity chromatography. This purification scheme achieved a nearly 250-fold purification of PCCase activity. However, throughout this purification of PCCase, ACCase copurified. Indeed, SDS-PAGE analysis of the final purified PCCase preparation identified two biotinylated polypeptides of about 240 and 230 kDa. These polypeptides have previously been described as subunits of ACCase (7). Furthermore, mixed substrate kinetic studies (8) with the purified PCCase/ACCase preparation indicated that the carboxylation of propionyl-CoA and acetyl-CoA were carried out by the same enzyme. Furthermore, both PCCase and ACCase activities were similarly affected by a variety of inhibitors. 

Carboxylation of propionyl-CoA leads to the formation of D(5)-methyl-malonyl-CoA. D-Methylmalonyl-CoA is converted by methylmalonyl-CoA racemase (EC 5.1.99.1) into the L(/ )-isomer which is in turn converted into succinyl-CoA by the vitamin B 12-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) (formerly isomerase), the succinyl-CoA subsequently entering the tricarboxylic acid cycle. 

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