Pyruvate reductive carboxylation

The reductive carboxylation of acetyl-CoA to pyruvate (Eq. 17-47) occurs only in a few types of bacteria. For most species, from microorganisms to animals, the oxidative decarboxylation of pyruvate to acetyl-CoA is irreversible. This fact has many important consequences. For example, carbohydrate... 

The dicarboxylate/4-hydroxybutyrate cycle starts from acetyl-CoA, which is reductively carboxylated to pyruvate. Pyruvate is converted to PEP and then car-boxylated to oxaloacetate. The latter is reduced to succinyl-CoA by the reactions of an incomplete reductive citric acid cycle. Succinyl-CoA is reduced to 4-hydroxybu-tyrate, the subsequent conversion of which into two acetyl-CoA molecules proceeds in the same way as in the 3-hydroxypropionate/4-hydroxybutyrate cycle. The cycle can be divided into part 1 transforming acetyl-CoA, one C02 and one bicarbonate to succinyl-CoA via pyruvate, PEP, and oxaloacetate, and part 2 converting succinyl-CoA via 4-hydroxybutyrate into two molecules of acetyl-CoA. This cycle was shown to function in Igrticoccus hospitalis, an anaerobic autotrophic hyperther-mophilic Archaeum (Desulfurococcales) [40]. Moreover, this pathway functions in Thermoproteus neutrophilus (Thermoproteales), where the reductive citric acid cycle was earlier assumed to operate, but was later disproved (W.H. Ramos-Vera et al., unpublished results). 

Apart from hydrogen evolution, the electrons of reduced ferredoxin can take alternative routes leading to biosynthesis. In anaerobic bacteria, reduced ferredoxin can be used directly for the reduction of pyridine nucleotides (Tagawa and Arnon (99) Valentine, Brill and Wolfe (107) Fredericks and Stadtman (44)) for the reduction of hydroxyla-mine to ammonia (Valentine, Mortenson, Mower, Jackson, and Wolfe (109) for COa fixation in the reductive carboxylation of acetyl-CoA to pyruvate (Bachofen, Buchanan, and Arnon (13) Raeburn and Rabino-witz (83) Andrews and Morris (3) Stern (98)) for the reduction of sulfite to sulfide (Akagi (1)) and, in the presence of ATP, it can be used for the reduction of N2 to NH3 (Mortenson (72,73) D Eustachio and Hardy (40)). The role of ferredoxin in these reactions as well as in the oxidative degradative reactions discussed above is summarized in Fig. 10. 

Evans, Buchanan, and Arnon (41a) have recently found that the ferredoxin-dependent pyruvate and a-ketoglutarate synthesizing reactions function in a new carbon cycle for the photosynthetic fixation of C02. The new cycle was named the reductive carboxylic acid cycle, and apart from pyruvate and a-ketoglutarate synthases, it includes certain of the enzymes associated with Krebs citric acid cycle, operating in the synthetic direction. Photoreduced ferredoxin and ATP, formed by photo-... 

The present studies confirm the earlier studies indicating the relatively great biosynthetic abilities of the methane bacteria and suggest that much of the cellular carbon compounds are probably synthesized from acetate and carbon dioxide. In view of the carbon dioxide and acetate requirements and the reductive carboxylation reactions shown to be involved in isoleucine synthesis in M. ruminantium (26) and the probability of similar carboxylation reactions in biosynthesis of isoleucine, alanine, and other amino acids in MOH, suggested by the studies on M. omelianskii (34), the operation of the pyruvate synthase reaction and some other reactions of the reductive carboxylic acid cycle (35, 36) as major pathways of biosynthesis of cellular materials in these bacteria is an attractive hypothesis. 

Similarly, the pyruvate (oxidase) dehydrogenase complex (PYOX) can be activated directly by electrogenerated methyl viologen radical cations (MV" ) as mediator. Thus, the naturally PYOX-catalyzed oxidative decarboxylation of pyruvic acid in the presence of coenzyme A (HSCoA) to give acetylcoenzyme A (acetyl-SCoA) (see section on oxidases) can be reversed. In this way, electroenzymatic reductive carboxylation of acetyl-SCoA is made possible (Fig. 15). 

Figure 15. Electroenzymatic reductive carboxylation of acetyl-SCoA to give pyruvate.

In the reductive carboxylic acid cycle of photosynthetic bacteria (92-94), which plays a role in the conversion of CO2 to precursors of fatty acids, amino acids, and porphyrins, four molecules of CO2 are fixed in one turn two of them are fixed to a-carbons of carbonyl groups of acetylcoenzyme A and succinyl-coenzyme A to generate pyruvate and a-keto glutarate, respectively ... 

Malic enzyme catalyzes the oxidative decarboxylation of malic acid or, in the reverse direction, the reductive carboxylation of pyruvic acid (26) [Eq. (8)]. 

Rhodospiriiium rubrum Rhodospiriiium tenue Reductive carboxylic acid cycle VI Pyruvate None... 

There are ADP-Glc PPases from organisms capable of having the Embden-Meyerhofif and Entner-Doudoroff pathways as well as anaerobic photosynthetic reductive carboxylic acid pathway. These ADP-Glc PPases are activated by three effectors Fru 1,6-bisP, Fru 6-P, and pyruvate. These three activators place them in class V (Table... 

Conversion of acetyl-CoA to triose phosphate (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) requires that two of the reactions of the catabolic pathway be replaced by alternative reactions catalysed by different enzymes these are (i) the pyruvate dehydrogenase complex-catalysed oxidative decarboxylation of pyruvate to acetyl-CoA, which is replac by the pyruvate synthase (EC 1.2.7.1)-catalysed reductive carboxylation of acetyl-CoA (eq. 3 I, Fig.), in which the reductant is reduced ferredoxin (Fd,ed). and (ii) the pyruvate kinase (EC 2.7.1.40)-catalysed, ATP-generating conversion of phosphoeno/pyruvate to pyruvate, which is replaced by the pyruvate, orthophosphate dikinase (EC 2.7.9.1)-catalysed, ATP-dtiven conversion of pyruvate to phosphoeno/pyruvate (eq. 4 F, Fig.),... 

Another reaction of lesser importance is the reductive carboxylation of pyruvate to malate. This is catalysed by the malic enzyme and the reducing power is supplied by NADPH. 

A variant of the above-mentioned carboxylation was found in the dicarboxylate/ 4-hydroxybutyrate pathway [39] (Scheme 9.6) where the acetyl-CoA is reductively carboxylated by pyruvate synthase at the expense of 2 equiv. of ferredoxin to afford pyruvate which is then phosphorylated. 

All reactions mediating fixation of CO by reductive carboxylation are readily reversible. Their equilibrium constant is sUghtly in favor of carboxylation, but at the low tensions of CO prevailing in cells and biological fluids the reverse action, i.e., oxidative decarboxylation, is favored unless appropriate mechanisms are broi t into play to displace the equilibrium position in favor of carboxylation. The two primary processes in each of these reactions, oxidation and decarboxylation or carboxylation and reduction, are intiinately interconnected and appear to be catalyzed by the same enzyme protein. The reactions to be considered in this section are the reductive car-boxylations of pyruvate, a-ketoglutarate, and ribulose 5-phosphate to L-malate, d-isocitrate, and 6-phosphogluconate, respectively. 

The key reaction was recognized by Utter to be the phosphorylation of oxaloacetate. Formation of the enol is facilitated in this compound and phosphoenol-pyruvate is then formed by phosphorylation with inosine triphosphate (ITP) (decarboxylation occurs at the same time). Oxaloacetate itself is provided by the direct carboxylation discussed above via a biotin enzyme. Alternately, the reductive carboxylation yields malate which is converted to oxaloacetate. The first reaction, although requiring additional ATP, appears to be the more important one, and, of course, the equilibrium favors C02-fixation more strongly in this way. The following... 

In all aerobic organisms, FA biosynthesis occurs via two different metabolic pathways (1) tricarboxylic acid cycle (TCA) or Krebs cycle and (2) reductive carboxylation pathway. Albert Szent-Gyorgyi (1893-1986, Hxmgary) discovered FA catalysis during his study (including on vitamin C) on cellular combustion process (TCA cycle) for which he was awarded the Noble Prize in Physiology or Medicine in 1937 (www.nobelprize.org). TCA involves CO2 fixation coupled with the conversion of pyruvate to oxaloacetate, the precursor to malate and fuma-rate (Fig. 8.3). Reductive CO2 fixation catalyzed by the enzyme pyruvate carboxylase xmder... 

A crystal structure of the C02 derivative of (8), K[Co(salen)( 71-C02)], haso been reported in which the Co—C bond is 1.99 A, the C—O bonds are both equivalent at 1.22 A and the O-C-O angle is 132°.125 Carboxylation of benzylic and allylic chlorides with C02 in THF-HMPA was achieved with (8) electrogenerated by controlled-potential electrolysis,126 in addition to reductive coupling of methyl pyruvate, diethyl ketomalonate and / -tolylcarbodiimide via C—C bond formation. Methyl pyruvate is transformed into diastereomeric tartrates concomitant with oxidation to the divalent Co(salen) and a free-radical mechanism is proposed involving the homolytic cleavage of the Co—C bond. However, reaction with diphenylketene (DPK) suggests an alternative pathway for the reductive coupling of C02-like compounds. 

The rapid rates of reduction of the oxalato (10) (k = 450 + 1,000 (H+)) and of the pyruvate (2) complexes (2A x 103at 25°C. and (H+) = 0.1) can hardly be understood as caused by chelation. Binoxalate does not chelate unless the proton is lost, and the rate law for the reduction of the complex shows that it brings a proton into the activated complex. Pyruvate almost certainly is not chelated in the product. Both groups are rapidly reduced by Craq.+2 when they are feee from the cobalt center. (The reduction of H2C2O4 by Craq+2 was explored by R. Milburn and the present author (29). The observations on pyruvate were made by R. Butler (2)). The complexes of pyridine-2-carboxylate and pyridine-4-carboxylate are rapidly reduced by Cr+2 at least in the forms which present the nitrogen without associated protons. Radical ion intermediates for these structures are not unreasonable. In fact, a stable free radical derived from AT-ethyl-4-carbethoxypyridinyl has been... 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

The question is therefore, what are the principal requirements of an autotrophic carbon-fixation mechanism An organic molecule serves as a C02 acceptor molecule, which becomes carboxylated by a carboxylase enzyme. This C02 acceptor molecule needs to be regenerated in a reductive autocatalytic cycle. The product that can be drained off from such a metabolic cycle should be a central cellular metabolite, from which all cellular building blocks for polymers can be derived examples of such central metabolites are acetyl-CoA, pyruvate, oxaloacetate, 2-oxoghitarate, phosphoe-nolpyruvate, and 3-phosphoglycerate. Importantly, the intermediates should not be toxic to the cell. The irreversible steps of the pathway are driven by ATP hydrolysis, while the reduction steps are driven by low-potential reduced coenzymes. 

The formation of /3-carbolines in vivo can be enhanced by injecting rats intraventricularly with tryptamine and pyruvic acid to produce significant brain levels of the 1-carboxylic acid TBC 38 (84). Injection of TBC 38 into rats resulted in the formation of DBC 34, TBC 29a, and BC 36 as the major metabolites (85). It was suggested that DBC 34 resulted from TBC 38 by oxidative decarboxylation and that TBC 29a was formed from DBC 34 by asymmetric reduction, with BC 36 occurring via further oxidation. The nonenzymatic decarboxylation of TBC 38 was greatly increased on addition of pydridoxal phosphate (86). The incubation of human platelets with tryptamine or serotonin afforded TBC 2S (87). These compounds were named tryptolines, which are identical with TBC, and were fully characterized by GC-MS techniques, and by comparison with synthetic standards and trifluoroacylated derivatives. 

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