TCA cycle, reverse

One of these alternate models, postulated by Gunter Wachtershanser, involves an archaic version of the TCA cycle running in the reverse (reductive) direction. Reversal of the TCA cycle results in assimilation of CO9 and fixation of carbon as shown. For each turn of the reversed cycle, two carbons are fixed in the formation of isocitrate and two more are fixed in the reductive transformation of acetyl-CoA to oxaloacetate. Thus, for every succinate that enters the reversed cycle, two succinates are returned, making the cycle highly antocatalytic. Because TCA cycle intermediates are involved in many biosynthetic pathways (see Section 20.13), a reversed TCA cycle would be a bountiful and broad source of metabolic substrates. 

The fourth view is a metabolism first view. Harold Morowitz points out that the reverse TCA cycle is autocatalytic (5). In a lecture at the Systems Chemistry conference in December 2006 in Barcelona, Albert Eschenmoser suggested that collectively autocatalytic (as defined just below) metabolic systems may be possible (6). 

Optimized redox potential in the fermentation broth has been revealed to give improved reducing power to shift the metabolic flux toward the reverse TCA cycle. This creates a higher ratio of succinate to by-products. Furthermore, it has also been exposed to enhance cell growth and the substrate consumption rate (Park and Zeikus, 1999 Li et al., 2010a,b). 

A reversed, reductive TCA cycle would require energy input to drive it. What might have been the thermodynamic driving force for such a cycle Wachtershanser hypothesizes that the anaerobic reaction of FeS and H9S to form insoluble FeS9 (pyrite, also known as fool s gold) in the prebiotic milieu could have been the driving reaction ... 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

The answer is that there is now a mechanism by which all of the other TCA cycle intermediates from oxaloacetate to sucdnyl CoA can be produced (all of these reactions are reversible). 

Wachtcrshauser s prime candidate for a carbon-fixing process driven by pyrite formation is the reductive citrate cycle (RCC) mentioned above. Expressed simply, the RCC is the reversal of the normal Krebs cycle (tricarboxylic acid cycle TCA cycle), which is referred to as the turntable of metabolism because of its vital importance for metabolism in living cells. The Krebs cycle, in simplified form, can be summarized as follows ... 

The term ketone bodies refers primarily to two compounds acetoacetate and P-hydroxy-butyrate, which are formed from acetyl-CoA when the supply of TCA-cycle intermediates is low, such as in periods of prolonged fasting. They can substitute for glucose in skeletal muscle, and, to some extent, in the brain. The first step in ketone body formation is the condensation of two molecules of acetyl-CoA in a reverse of the thiolase reaction. 

Assay techniques GDH utilizes both nicotinamide nucleotide cofactors NAD+ in the direction of N liberation (catabolic) and NADP+ for N incorporation (assimilatory). In the forward reaction, GDH catalyzes the synthesis of amino acids from free ammonium and Qt-kg. The reverse reaction links amino acid metabolism with TCA cycle activity. In the reverse reaction, GDH provides an oxidizable carbon source used for the production of energy as weU as a reduced electron carrier, NADH, and production of NH4+. As for other enzymes, spectrophotmetric methods have been developed for measuring oxoglutarate and aminotransferase activities by assaying substrates and products of the GDH catalyzed reaction (Ahmad and Hellebust, 1989). 

All microbes, including chemoorganohetero-trophs, possess some ability to engage in reversible carboxylation (i.e., CO2-C assimilation into an organic compound) and decarboxylation reactions, some of which lead to the incorporation of a significant amount of CO2 (Wood, 1985). Here, we briefly consider the biochemical pathways that photo- and chemolithotrophic bacteria deploy in order to produce the majority of their biomass. The four major C02-fixing pathways are the Calvin cycle, the acetyl-CoA pathway, the reductive tricarboxylic acid (TCA) cycle, and the 3-hydroxypriopionate cycle. 

However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity. 

Fumarase is an enzyme component of the TCA cycle that catalyzes the reversible reaction of fumarate to L-malate with equilibrium favoring malate production. It is a soluble enzyme with high turnover number. In one report, fumarate content in some organisms can be as high as lOOOmg/kg of wet cells [80]. Theoretically, a malate weight yield of 115% can be obtained from fumarate. However, in reality, a weight yield of 90-95% is often obtained. 

Fig. 2. Metabolic interactions between neurons and astrocytes. Glucose enters the brain through the astrocytic end feet that envelop brain capillaries. In the astrocytes some of the glucose is metabolized to lactate which is exported to the extracellular fluid and taken up by neurons. In neurons lactate is converted to pyruvate which is either decarboxyiated to acetyl-CoA or carboxykited to malate to enter the TCA cycle. Glutamate may therefore be formed in neurons from a-ketoglutarate or from glutamine, which is imported from astrocytes. The glutamate that is released is taken up by astrocytes and amidated to glutamine or metabolized via the TCA cycle. The malate thus formed may leave the TCA cycle and become decarboxyiated to pyruvate and lactate. For lack of space, astrocytic pyruvate carboxylation is indicated only by the reversible formation of lactate. Notice that the relative importance of the various pathways in vivo is a matter of debate (see text).

F. 20.19. Major anaplerotic pathways of the TCA cycle. 1 and 3 (blue arrows) are the two major anabohc pathways. (1) Pyruvate carboxylase (2) Glutamate is reversibly converted to a-ketoglutarate by transaminases (TA) and glutamate dehydrogenase (GDH) in many tissues. (3) The carbon skeletons of valine and isoleucine, a 3-carbon unit from odd chain fatty acid oxidation, and a number of other comprounds enter the TCA cycle at the level of succinyl CoA. Other amino acids are also degraded to fumarate (4) and oxaloacetate (5), principally in the liver. 

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