PEPCK cytosolic

Phosphoenolpyruvate carboxykinase (PEPCK) deficiency is distinctly rare and even more devastating clinically than deficiencies of glucose-6-phosphatase or fructose-1,6-bisphosphatase. PEPCK activity is almost equally distributed between a cytosolic form and a mitochondrial form. These two forms have similar molecular weights but differ by their kinetic and immunochemical properties. The cytosolic activity is responsive to fasting and various hormonal stimuli. Hypoglycemia is severe and intractable in the absence of PEPCK [12]. A young child with cytosolic PEPCK deficiency had severe cerebral atrophy, optic atrophy and fatty infiltration of liver and kidney. 

The pathway of glucose breakdown is indicated in Figure 4.25 by heavy dots. Steps that are Inactivated by a rise in the glucagon/Insulin ratio are indicated by boldface Xs. As explained, PEPCK (more accurately, cytosolic PEPCK) activity is controlled by cellular levels of cAMP in the following manner. Cyclic AMP combines with a special protein (CREBP) to form a complex that binds to DN A. Where does the cAMP/CREBP complex bind The complex binds to a short stretch of... 

A species specificity mi ht be pointed out with respect to the location of PE PC K, an enzyme used for gluconeogenesis, OAA is converted to PEP by PEPCK in the liver Cytoplasm of rats and mice because PEPCK is a cytosolic enzyme in these animals. In the livers of chickens and rabbits, however, OAA is converted to PEP in the mitochondria, because PEKK is mitochondrial in these animals, PEP must then be transported to the cytosol. PEPCK is evenly distributed between the mitochondria and the cytosol in human and cow livers (Pilkis et itf., 1988). 

In humans, oxaloacetate must be transported out of the mitochondrion to supply the cytosolic PEPCK. Because there is no mitochondrial carrier for oxaloacetate and its diffusion across the mitochondrial membrane is slow, it is transported as malate or asparate (Figure 15-2). The malate shuttle carries oxaloacetate and reducing equivalents, whereas the aspartate shuttle, which does not require a preliminary reduction step, depends on the availability of glutamate and a-ketoglutarate in excess of tricarboxylic acid (TCA) cycle requirements. 

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). 

Short-term regulation of this reaction is accomplished by changes in the relative proportions of substrates and products. Increased concentrations of oxaloacetate and GTP (or ITP) increase the rate, and accumulation of phosphoenolpyruvate and GDP (or IDP) decreases it. The cytosolic PEPCK is also under long-term regulation by hormones. Its synthesis is increased by corticosteroids. Starvation and diabetes mellitus increase the synthesis of cytosolic PEPCK, whereas refeeding and insulin have the opposite effect. 

The increase in glutamate favors transamination of oxaloacetate and limits oxaloacetate availability for phosphoenolpyruvate synthesis. When the [NADH]/[NAD+] ratio is low, malate formation occurs more readily. The cytosolic PEPCK is relatively unaffected by the mitochondrial [NADH]/[NAD+] ratio. Once malate and aspartate are transported to the cytosol and they are reconverted to oxaloacetate, cytosolic PEPCK can convert it to phosphoenolpyruvate. 

Oxaloacetate, produced from malate or aspartate in the cytosol, is converted to PEP by the cytosolic PEPCK (see Eig. 31.7A). 

Phosphoenolpyruvate carboxykinase is induced. Oxaloacetate produces PEP in a reaction catalyzed by PEPCK. Cytosolic PEPCK is an inducible enzyme, which means that the quantity of the enzyme in the cell increases because of increased transcription of its gene and increased translation of its mRNA. The major inducer is cyclic adenosine monophosphate (cAMP), which is increased by hormones that activate adenylate cyclase. Adenylate cyclase produces cAMP from ATP. Glucagon is the hormone that causes cAMP to rise during fasting, whereas epinephrine acts during exercise or stress. cAMP activates protein kinase A, which phosphorylates a set of specific transcription factors (CREB) that stimulate transcription of the PEPCK gene (see Chapter 16 and Pig. 16.18). Increased synthesis of mRNA for PEPCK results in increased synthesis of the enzyme. Cortisol, the major human glucocorticoid, also induces PEPCK. 

Oxaloacetate is decarboxylated and phosphorylated in the cytosol by PEP-carboxykinase (also referred to as PEPCK) The reaction is... 

Figure 10-4. Postnatal changes in newborn rat liver cytosolic phosphoenolopyruvate carboxykinase (PEPCK) activity and gluconeogenic rate in vivo from [14C-lactate]. Reprinted with permission from Girard et al. (1992).

The glucocorticoid cortisol is secreted from the adrenal cortex as a stress response under the control of adrenocorticotropic hormone (ACTH, corticotropin) produced by the anterior pituitary. Cortisol promotes catabolism by inducing synthesis of specific proteins. Cortisol binds to a cytosolic cortisol receptor which then translocates to the nucleus and switches on the expression of specific genes, notably that for PEP carboxykinase (PEPCK). Cortisol-induced expression of the key gluconeogenesis enzyme PEPCK increases levels of the enzyme and hence increases gluconeogenesis and available blood glucose. The cAMP-and cortisol-mediated pathways for induction of PEPCK expression are further linked by CREB-dependent expression of a coactivator protein PGC-1 that promotes cortisol-dependent expression of PEPCK. 

Pyruvate carboxylase is a mitochondrial enzyme in animal cells, whereas PEPCK is almost exclusively mitochondrial in some species (e.g., pigeons) and cytosolic in others (e.g., rats and mice). In humans (and guinea pigs), PEPCK occurs in both mitochondria and cytosol. An interesting consequence of this species differences is that quinolinate, an inhibitor of cytoplasmic PEPCK, causes hypoglycemia in rats but is less active in humans and guinea pigs. 

The CO2 that was added to pyruvate to form oxaloacetate is released in the reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), which generates PEP (Fig. 31.7A). For this reaction, GTP provides a source of energy as well as the phosphate group of PEP. Pyruvate carboxylase is found in mitochondria. In various species, PEPCK is located either in the cytosol or in mitochondria, or it is distributed between these two compartments. In humans, the enzyme is distributed about equally in each compartment. 

Oxaloacetate, generated from pyruvate by pyruvate carboxylase or from amino acids that form intermediates of the TCA cycle, does not readily cross the mitochondrial membrane. It is either decarboxylated to form PEP by the mitochondrial PEPCK or it is converted to malate or aspartate (see Figs. 31.7B and 31.7C). The conversion of oxaloacetate to malate requires NADH. PEP, malate, and aspartate can be transported into the cytosol. 

The metabolic steps in gluconeogenesis occur in two intracellular compartments (Fig. 3.2) the cytosol and the mitochondrial matrix. The enzymes of the tricarboxylic acid cycle reside in the mitochondrial matrix, apart from succinate dehydrogenase which is present in the inner mitochondrial membrane, whereas most of the enzymes of the gluconeogenic pathway are present in the cytosol. Transaminases, such as alanine aminotransferase and aspartate aminotransferase, are present both in mitochondria and cytosol of the domestic fowl liver (Sarkar, 1977). One of the control enzymes in gluconeogenesis, PEPCK, has a different intracellular distribution in avian liver compared with mammalian liver (Table 3.3). PEPCK in both pigeon and domestic fowl liver is present almost exclusively (> 99%) in mitochondria (Soling et al.. 1973), whereas in most mammals that have been studied, it is present mainly in the cytosol, and only present, if at all, in smaller amounts in... 

PEPCK (absent from muscle) Kidney Cytosol monomer 75 kDa Mn -" Mainly pyruvate and amino... 

Oxaloacetate is formed from pyruvate by pyruvate carboxylase, located in the mitochondria. Pyruvate carboxylase is a biotin-requiring enzyme its kinetic mechanism has been studied in detail using the enzyme purified from domestic fowl liver (Attwood Graneri, 1992). In tissues where PEPCK is located in the cytosol, its substrate oxaloacetate is required in the cytosol for the formation of PEP for gluconeogenesis. The malate-aspartate shuttle is required for gluconeogenesis in avian kidney, according to the scheme in Fig. 3.3, but whether or not it is also required for gluconeogenesis in avian liver is unresolved. There is evidence for the existence of a... 

Phosphoenolpyruvate carboxykinase (PEPCK) in the cytosol needs GTP, is stimulated by glucagon and inhibited by insulin. 

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