Pyruvate carboxylase substrate cycle

Substrate availability for certain reactions can be optimized by anaplerotic ( topping-up ) reactions. For example, citrate synthase is a key control point of the TCA cycle. The co-substrates of citrate synthase are acetyl-CoA and oxaloacetate (OAA) and clearly, restriction in the availability of either substrate will decrease the rate of the citrate synthase reaction. Suppose, for example, a situation arises when acetyl-CoA concentration is significantly higher than that of OAA, the concentration of the latter can be topped-up and the concentration of acetyl-CoA simultaneously reduced by diverting some of the pyruvate away from acetyl-CoA synthesis (via pyruvate dehydrogenase) to OAA synthesis (via pyruvate carboxylase) as shown in Figure 3.1. The net effect is to balance the relative concentrations of the two co-substrates and thus to promote citrate synthase activity. 

Where two enzymes compete for the same substrate, we expect to see some form of metabolic control and in this case the concentrations of NADH and acetyl-CoA are the key controlling factors (Figure 6.44). When glucose is not available as a fuel, metabolism switches to 3- oxidation of fatty acids, which generates more than sufficient quantities of both NADH and acetyl-CoA to drive the TCA cycle and to maintain oxidative phosphorylation. Pyruvate dehydrogenase activity is suppressed and pyruvate carboxylase is stimulated by ATP, NADH and acetyl-CoA (strictly speaking by low mitochondrial ratios of ADP/ATP, NAD+/NADH and coenzyme A/acetyl-CoA), so... 

As we have seen, normally pyruvate would be the substrate for pyruvate dehydrogenase complex to form acetyl-CoA, but during fasting in the absence of glucose, acetyl -CoA for the TCA cycle is derived from fatty acid (3-oxidation (see Section 7.5.2) so pyruvate is diverted into oxaloacetate by the enzyme pyruvate carboxylase. Thus any amino acids whose carbon skeletons can be converted into pyruvate, OAA or another substrate of the TCA cycle, can be used for glucose synthesis. 

Figure 9.24 Control of the oxaloacetate concentration and hence the flux through the cycle by pyruvate carboxylase. The activity of pyruvate carboxylase is increased by an increase in its substrate, pyruvate, and its allosteric regulator, acetyl-CoA. Regulation of the activity is important to increase the concentration of oxaloacetate which increases the flux through the cycle. An increase in the rate of glycolysis increases the concentration of pyruvate, and an increase in the rate of fatty acid oxidation increases that of acetyl-CoA. Both result in an increase in the concentration of oxaloacetate and hence in the flux through the cycle, providing coordination between the rates of glycolysis, fatty acid oxidation and the cycle.

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. 

Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction (see Figure 8.24). This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis (see p. 116). 

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two-step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO2, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate. 

The product of this reaction, oxaloacetate, can either enter the gluconeogenic pathway (Chap. 11) by way of malate or condense with acetyl-CoA to yield citrate. Pyruvate carboxylase is an allosteric enzyme, and it is activated by the heterotropic effector, acetyl-CoA. Thus, pyruvate in the mitochondria is the substrate for either pyruvate dehydrogenase or pyruvate carboxylase, the activities of which, in turn, are controlled by reactants associated with the citric acid cycle. The interplay among pyruvate dehydrogenase, pyruvate carboxylase, pyruvate, and the citric acid cycle is shown in Fig. 12-9. 

In liver, most of the phosphoenolpyruvate is formed by the continued action of pyruvate carboxykinase and pyruvate carboxylase. In fact, the carboxykinase reaction determines whether the original substrate, oxaloacetate, is converted to glucose or oxidized in the Krebs cycle. The conversion of pyruvic acid to phosphoenolpyruvate is a rate-limiting step in the conversion of 3-carbon compounds (alanine and lactic acid, for example) to glucose. When glucogenesis is increased e.g./ n diabetes and after the administration of glucocorticoids), carboxylase and carboxykinase activities are also increased. 

For glutamate production, a balanced supply of acetyl-CoA and oxaloacetate is important to proceed TCA cycle reactions toward 2-oxoglutarate, because it is a substrate for glutamate biosynthesis catalyzed by GDH. In particular, anaplerotic reactions, which supply oxaloacetate from the glycolytic intermediates, are caudal. C. glutamicum possesses two enzymes for anaplerotic reactions phosphoenolpyru-vate carboxylase (PEPC) encoded by ppc and pyruvate carboxylase (PC) encoded by pyc (Bormann et al. 1992 Eikmanns et al. 1995 O Regan et al. 1989 Peters-Wendisch et al. 1998). PC requires biotin as a cofactor for its activity. MFA was conducted to understand the role of these anaplerotic reactions in glutamate production. 

Biotin is the coenzyme required by enzymes that catalyze carboxylation of a carbon adjacent to a carbonyl group. For example, pymvate carboxylase converts pyruvate—the end product of carbohydrate metabolism—to oxaloacetate, a citric acid cycle intermediate (Figure 25.2). Acetyl-CoA carboxylase converts acetyl-CoA into malonyl-CoA, one of the reactions in the anabolic pathway that converts acetyl-CoA into fatty acids (Section 19.21). Biotin-requiring enzymes use bicarbonate (HCOs ) for the source of the carboxyl group that becomes attached to the substrate. 

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