Succinyl CoA formation

Gibson et al. [35a] were the first to demonstrate that succinyl-CoA was one of the substrates of ALA-synthetase. In blood erythrocytes containing mitochondria and in fiver cells the main pathway for succinyl-CoA formation appears to be via the enzyme complex a-KG oxidase and the citric acid cycle. The oxidation of a-KG requires the action of five enzymes, each with a different vitamin as prosthetic... 

Another reaction for the formation of succinyl-CoA is the carboxy-lation of propionyl-CoA (biotin enzyme) to methyl malonyl-CoA, and the conversion of methyl malonyl-CoA by a soluble vitamin B12 enzyme to succinyl-CoA. These enzymes provide reactions for the incorporation of propionic acid into the citric acid cycle the reactions are especially important in ruminants in which large amounts of propionic acid are formed by the bacteria in the rumen. The significance of this method of succinyl-CoA formation for the synthesis of ALA in fiver or red blood cell [Nakao and Takaku, 51b] is not known. This method is not used by higher plants, for they do not contain vitamin B12. 

The free energy of the transfer reaction is a function of pH, because in addition to the carboxyl groups the carbonyl group of acetoacetate thioester can ionize (as an enol). Since this ionization is favored by alkaline media, the equilibrium is somewhat shifted towards acetoacetyl CoA formation at higher pH values. The reaction favors succinyl CoA formation at all pH values studied, however (Fo at pH 7.0 = +3470 cal.). Another factor that influences the apparent equilibrium of the transferase is the presence of Mg++. This ion forms a complex with the /3-ketothioester. The enzyme has been assayed by measuring the appearance of the absorption band of the j8-ketothioester at 303 m/ , where simple thioesters do not absorb. This band is shifted slightly and the extinction coefficient greatly increased by Mg++, which has therefore been added to assay systems to increase the sensitivity. CoA transferase does not require Mg" for activity, however. 

In erythrocytes the main pathway for succinyl CoA formation appears to be from a-ketoglutaric acid by oxidation via the citric acid cycle, through TPP and lipoic acid, to form succinyl lipoate which with CoA forms succinyl CoA. The conversion of succinate to succinyl CoA has been shown by Shemin and Kumin (38) who used labeled succinate in the presence of malonate. 

D. Coenzyme A.—Succinyl phosphate (42) reacts rapidly and non-enzymatically with CoA in the pH range 3—8 to yield succinyl CoA (43). This reaction is dependent on the presence of a suitably situated free carboxy-group as such nucleophilic attack at carbon is not known with other acyl phosphates. Moreover, maleyl phosphate reacts rapidly with CoA while fumaryl phosphate fails to react under the same conditions. Hence the formation of a cyclic intermediate (44) from succinyl phosphate is... 

The next step, the formation of succinyl CoA, also involves one oxidation and one decarboxylation. it is catalyzed by 2-oxoglutarate dehydrogenase, a multienzyme complex closely resembling the PDH complex (see... 

In animal metabolism, derivatives of cobalamine are mainly involved in rearrangement reactions. For example, they act as coenzymes in the conversion of methylmalonyl-CoA to succinyl-CoA (see p. 166), and in the formation of methionine from homocysteine (see p. 418). In prokaryotes, cobalamine derivatives also play a part in the reduction of ribonucleotides. 

Kavanaugh-Black, A. Connolly, D.M. Chugani, S.A. Chakrabarty, M. Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa complex formation with succinyl-CoA synthetase. Proc. Natl. Acad. Sci. USA, 91, 5883-5887 (1994)... 

The next step is another oxidative decarboxylation, in which a-ketoglutarate is converted to succinyl-CoA and C02 by the action of the a-ketoglutarate dehydrogenase complex NAD+ serves as electron acceptor and CoA as the carrier of the succinyl group. The energy of oxidation of a-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA ... 

The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of a-ketoglutarate is a substrate-level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase (see Fig. 14-2). The GTP formed by succinyl-CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase (p. 505) ... 

Although the citric acid cycle directly generates only one ATP per turn (in the conversion of succinyl-CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain via NADH and FADH2 and thus lead to formation of a large number of ATP molecules during oxidative phosphorylation. 

Propionyl-CoA is first carboxylated to form the d stereoisomer of methylmalonyl-CoA (Pig. 17—11) by propionyl-CoA carboxylase, which contains the cofactor biotin. In this enzymatic reaction, as in the pyruvate carboxylase reaction (see Pig. 16-16), C02 (or its hydrated ion, HCO ) is activated by attachment to biotin before its transfer to the substrate, in this case the propionate moiety. Formation of the carboxybiotin intermediate requires energy, which is provided by the cleavage of ATP to ADP and Pi- The D-methylmalonyl-CoA thus formed is enzymatically epimerized to its l stereoisomer by methylmalonyl-CoA epimerase (Pig. 17-11). The L-methylmal onyl -CoA then undergoes an intramolecular rearrangement to form succinyl-CoA, which can enter the citric acid cycle. This rearrangement is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5 -deoxyadenosyl-cobalamin, or coenzyme Bi2, which is derived from vitamin B12 (cobalamin). Box 17—2 describes the role of coenzyme B12 in this remarkable exchange reaction. 

Formation of S-aminolevulinic acid (ALA) All the carbon and nitrogen atoms of the porphyrin molecule are provided by two simple building blocks glycine (a nonessential amino acid) and succinyl CoA (an intermediate in the citric acid cycle). Glycine and succinyl CoA condense to form ALA in a reaction catalyzed by ALA synthase (Figure 21.3) This reaction requires pyridoxal phosphate as a coenzyme, and is the rate-controlling step in hepatic porphyrin biosynthesis. 

The thioester bond makes succinyl-CoA an activated intermediate. Although some succinyl-CoA is used in the synthesis of heme in animals, most of it is retained in the TCA cycle where it leads to the regeneration of the oxaloacetate needed to keep the cycle operating. The thioester is converted to succinate in a coupled reaction that results in the formation of GTP. 

Early isotope tracer experiments by David Shemin permitted the elucidation of the formation of the immediate precursor of the porphyrin needed for the cytochromes and for hemoglobin. These studies indicated that the glycine methylene carbon and nitrogen were incorporated along with both carbons of acetate. Subsequent enzymatic studies in both bacteria and animals revealed a condensation reaction between succinyl-CoA and glycine to yield 5-amino-levulinate and C02 (presumably by way of an enzyme-bound /3-keto acid, a-amino-/3-ketoadipate) (fig. 22.13). 

Figure 3.7 General scheme representing the strategy used by four different carbon dioxide-fixation pathways [40], These pathways have in common the formation of succinyl-CoA from acetyl-CoA and two inorganic carbons. The vertical arrows point to the C02-fixation products released from these...

ALA synthase is a pyridoxal phosphate-dependent enzyme and promotes Schiff-base formation between its coenzyme and glycine (67 in Fig. 37). Nucleophilicity at C-2 of the glycine could be generated either by decarboxylation or by abstraction of a proton. In the first case 5-aminolaevulinic acid would retain both methylene protons of glycine, in the second, one of the protons would be lost to the medium (Fig. 37). Acylation of the pyridoxal-bound intermediate (68 or 69) by succinyl-CoA would constitute the next step and this could be followed either by direct hydrolysis of the Schiff-base or by decarboxylation with subsequent hydrolysis depending on which course was chosen in the first stage of the reaction. 

This mitochondrial enzyme (ALA synthetase) catalyzes the formation of 6-aminolevulinic acid (ALA) from glycine and succinyl-CoA. This is the initial step in heme biosynthesis. 

Figure 9.78 HPLC-based assay for sucdnyl-CoA synthetase from rabbit liver mitochondria. One hundred microliters of diluted supemate was added to 3.0 mL containing 50 mAf succinate, 33 mAf Na-Hepes, pH 7.4, 5 mAf MgCl2,1 mAf CoA, 1 mAf GTP, and 5 jug of oligomycin. The reaction was run at 30°C and aliquots of 0.30 mL were removed at 1-minute intervals and transferred to autosampler vials containing 0.2 mL of 0.2 Af formic acid. Nucleotides were separated by HPLC. The UV detector was set to 254 nm. The chromatographic profiles for the reaction after 0, 5, and 10 minutes are shown. Peaks (top profile) 1, GMP 2, GDP 3, GTP 4, CoA and 5, succinyl-CoA. (A) Linearity of succinyl-CoA (S-CoA) formation, expressed as the percentage conversion of CoA to sucdnyl-CoA, with time. (B) Linearity of S-CoA formation with micrograms of protein added for 5 minute assays carried out in a volume of 0.30 mL. The latter assays were carried out in duplicate. The values shown are averages of the duplicate assays. (From Lambeth and Muhonen, 1993.)...

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