Pyruvate carboxylase stimulation

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... 

The FADHj and NADH are oxidized in the electron transport chain, providing ATP. In musde and adipose tissue, the acetyl CoA enters the citric acid cyde. In liver, the ATP may be used for gluconeogenesis, and the acetyl CoA (which cannot be converted to glucose) stimulates gluco-neogenesis by activating pyruvate carboxylase. 

Pyruvate carboxylase is stimulated by acetyl-CoA, increasing the rate of gluconeogenesis when the cell already has adequate supplies of other substrates (fatty acids) for energy production. 

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction. 

Acetyl-CoA is a critical regulator of the fate of pyruvate it allosterically inhibits pyruvate dehydrogenase and stimulates pyruvate carboxylase (see Fig. 15-20). In these ways acetyl-CoA prevents it own further production from pyruvate while stimulating the conversion of pyruvate to oxaloacetate, the first step in gluconeo-genesis. 

Answer Fatty acid catabolism increases the level of acetyl-CoA, which stimulates pyruvate carboxylase. The resulting increase in oxaloacetate concentration stimulates acetyl-CoA consumption through the citric acid cycle, causing the citrate and ATP concentrations to rise. These metabolites inhibit glycolysis at PFK-1 and inhibit pyruvate dehydrogenase, effectively slowing the utilization of sugars and pyruvate. 

There have been reports of other glucagon actions in the liver which can be related to the elevation of cAMP, but whose molecular mechanisms are not well defined. Examples are the stimulations of ketogenesis, ureogenesis, amino acid transport, respiration and ion fluxes, the rapid changes in pyruvate dehydrogenase and pyruvate carboxylase, and the induction of P-enolpyruvate carboxykinase and other enzymes. 

Pyruvate carboxylase also requires a monovalent cation for activity. The activity of the purified enzyme was measured in the presence of various monovalent cations, as indicated in Table 10.7. Similar patterns of stimulation have been fovmd for acetyl-CoA synthetase, an enzyme used in acetate metabolism (Webster, 1966), propionyl-CoA carboxylase (Giorgio and Plaut, 1967), and several other enzymes (Suelter, 1970). Maximal activity of the aforementioned enzymes usually occurs at a wide range of potassium concentrations, that is, from 50 to 150 mM. There is therefore little reason to believe that the slight changes in intracellular K concentrations that can occur under normal conditions or during K deficiency result in an impairment in the activities of these enzymes or in some t)q>e of regulation of the activities. 

By decreasing the mitoehondrial concentration of glutamate, an inhibitor of pyruvate carboxylase, through stimulation of the TCA cycle (secondary to the increase in mitochondrial acetyl-CoA) and the aspartate shuttle (secondary to the increase in cytosolic PEPCK induced by glucagon). 

A large intracellular pool of a) ions (caused by a negative effect of severe limitation of b) on protein turnover) and an increased respiratory activity, which in part is not coupled to c) synthesis, stimulates metabolic flux throu glycolysis without significant metabolic control. This, togettier with d) pyruvate carboxylase and the peculiarities in the operation of the TCA-cyde, results in elevat cellular concentrations of e>. This in turn enhances dtric add accumulation by inhibiting i) dehydrogenase. 

The activation of pyruvate carboxylase by acetyl CoA is a sigmoidal response, with the steep part of the curve between the normal low and high ranges of acetyl CoA. Thus, as acetyl CoA increases in concentration, there is a marked stimulation of pyruvate carboxylase (Fig. 13.3). 

For PEPCK to function in gluconeogenesis, oxaloacetate produced in the pyruvate carboxylase reaction in the mitochondria, must be transported to the cytoplasm. PEPCK is not under any known allosteric control. Activity of the enzyme is regulated by hormonal control of its transcription. Glucagon stimulates transcription of the structural gene for PEPCK. Insulin inhibits transcription of the enzyme. By inhibiting PEPCK gene transcription, insulin tends to depress gluconeogenesis rates. 

Pyruvate carboxylase - This enzyme is found only in the mitochondrial matrix, apart from the other enzymes of glycolysis and gluconeogenesis. It can be activated by acetyl-CoA, but it is not clear what role it has in the overall control of the enzyme, since cellular levels of acetyl-CoA are far higher, under most conditions, than the concentration giving half-maximal stimulation. 

Gluconeogenesis in liver is strongly promoted by glucagon and adrenaline. The effects, mediated by cAMP, include stimulation of fructose 1,6-bisphospha-tase and inhibition of phosphofructo-l-kinase, both caused by the drop in the level of fructose The conversion of pyruvate to PEP via oxaloacetate is also promoted by glucagon. This occurs primarily by stimulation of pyruvate carboxylase (Eq. 

EXAMPLE 13.13 Pyruvate carboxylase equips a cell to produce more of the key carrier metabolite oxaloacetate for use in the Krebs cycle. Even though oxaloacetate is recycled, a low concentration potentially limits the rate of acetyl-CoA oxidation. In strenuous exercise, flux within the Krebs cycle is increased by the addition of the amount of oxaloacetate via this filling or anaplerotic reaction (Greek ana, again pleros, to fill). Pyruvate carboxylase is stimulated by a rise in acetyl CoA concentration in the cell, and this occurs when the rate of its production exceeds its loss by oxidation. 

The availability of oxaloacetate, which acts as the metabolite carrier in the Krebs cycle, may also be limited. The production of oxaloacetate is achieved by acetyl-CoA-induced stimulation of pyruvate carboxylase (Sec. 11.8 Fig. 11-17). 

The effect of manganese deficiency or excess on carbohydrate homeostasis have been studied by Keen, Hmley and coworkers, with some focus on pyruvate carboxylase and PEP carboxykinase [302-304]. Other evidence indicates that Mn(II) has an insulin-mimetic effect, acting to stimulate protein kinases or phosphatases that control enzymes involved in glycolysis, gluconeogenesis, or the hexose mono-P pathway [305-307]. A key role of Mn(II) in mucopolysaccharide metabolism has long been recognized [6]. 

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