- Pyruvate carboxylase
Pyruvate carboxylase is an
enzymeof the ligaseclass that catalyzesthe irreversible carboxylationof pyruvateto form oxaloacetate(OAA).
It is the important anaplerotic reaction that provides oxaloacetate precursor for the
citric acid cycleduring the time of exercise. The enzyme is a mitochondrial proteincontaining a biotinprosthetic group, requiring magnesiumor manganeseand acetyl CoA, and occurs in liverbut not in muscle. It is an ATP-dependent enzyme. High levels of ADP inhibit phosphorylation of the enzyme thereby keeping the enzyme active, while acetyl-CoAacts as an allosteric activator of the enzyme [Scrutton MC, Utter MF (1967) Pyruvate carboxylase. IX. Some properties of the activation by certain acyl derivatives of coenzyme A. J. Biol Chem. 242:1723-1735.] .
Pyruvate carboxylase was first discovered in 1959 by M. F. Utter and D. B. Keech [Cohen ND, Beegen H, Utter MR, Wrigley NG. J Biol Chem. Mar 10;254(5):1740-7 (1979). A re-examination of the electron microscopic appearance of pyruvate carboxylase from chicken liver.] . Since then it has been found in a wide variety of prokaryotes and eukaryotes including fungi, bacteria and plants as well as higher organisms [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] . In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose induced insulin secretion by pancreatic islets. Oxaloacetate produced by PC is an important intermediate which is used in these biosynthetic pathways [Jitrapakdee, S. et al. Biochem and Biophy Comm. 295; 387–393 (2002).] . In mammals, PC is expressed in a tissue-specific manner, with its activity found to be highest in the liver and kidney (gluconeogenic tissues), in adipose tissue and lactating mammary gland (lipogenic tissues), and in pancreatic islets. Activity is moderate in brain, heart and adrenal gland, and least in white blood cells and skin fibroblasts [Jitrapakdee, S., et al. Biochem Journal 223, 695–700 (1996). Identification of Novel Alternatively Spliced Pyruvate Carboxylase mRNAs with Divergent 59-Untranslated Regions Which Are Expressed in a Tissue-Specific Manner.] . The roles of PC in these different tissues are described below.
Pathways for Glucose Synthesis (
It is also the first pathway for the synthesis of
phosphoenolpyruvatefrom pyruvate. Pyruvate is first converted by pyruvate carboxylase to oxaloacetatein the mitochondrion. During the reaction, one molecule of ATP is hydrolysed. The OAA is then decarboxylated and simultaneously phosphorylated, which is catalyzed by PEP carboxykinase to produce PEP in the cytosol. Transport of OAA from the mitochondrion to the cytosol is mediated by the malate/OAA shuttle.
Structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains a single biotin moiety acting as a swinging arm to convey CO2 between two catalytic subsites. Each subunit contains three functional domains: the biotin carboxylation (BC) domain, the transcarboxylation (CT) domain and the biotin carboxyl carrier (BCCP) domain [Kondo, S. et al. Acta Crystallogr. D 60, 486 (2004). Structure of the biotin carboxylase subunit of pyruvate carboxylase. ] .
Pyruvate carboxylase uses a covalently attached biotin cofactor to catalyze the ATP– dependent carboxylation of pyruvate to oxaloacetate in two steps. Biotin is initially carboxylated at the BC active site by ATP and bicarbonate. The carboxyl group is subsequently transferred by carboxybiotin to a second active site in the CT domain, where pyruvate is carboxylated to generate oxaloacetate. The BCCP domain transfers the tethered cofactor between the two remote active sites. The allosteric binding site in PC offers a target for modifiers of activity that may be useful in the treatment of obesity or type II diabetes, and the mechanistic insights gained from the complete structural description of RePC (R. etli) permit detailed investigations into the individual catalytic and regulatory sites of the enzyme [Martin St. Maurice, et al. Science 317, 1076 (2007). Domain Architecture of Pyruvate Carboxylase, a Biotin-Dependent Multifunctional Enzyme.] .
The reaction mechanism can be subdivided into two partial reactions. In the first partial reaction, biotin is carboxylated using ATP and HCO3- as substrates [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] . In most species, this reaction requires acetyl-CoA as an allosteric activator. The second partial reaction involves the transfer of CO2 to the acceptor molecule, pyruvate, to form oxaloacetate. The major regulator of enzyme activity, acetyl CoA, stimulates the cleavage of ATP in the first partial reaction and also it has been shown to induce a conformational change in the tetrameric structure of the enzyme [Jitrapakdee, S. et al. Biochem and Biophy Comm. 295; 387–393 (2002).] .
Role in Gluconeogenesis
Due to the limited amount of glycogen stored in the liver, additional glucose must be synthesized by gluconeogenesis. Pyruvate carboxylase is essential in the process of gluconeogenesis, catalyzing the ATP-dependent fixation of carbon dioxide (carboxylation) to pyruvate to form oxaloacetate. This process occurs in the liver and kidney. In fasting conditions, gluconeogenesis accounts for up to 96%of total glucose production. The presence of very high PC activity, together with high activities of other gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase and glucose-6-phosphatase in liver and kidney cortex, suggests that a primary role of PC is to participate in gluconeogenesis in these tissues. During fasting or starvation when endogenous glucose is required for certain tissues (brain, white blood cells and kidney medulla), expression of PC and other gluconeogenic enzymes has been shown to be elevated [Rothman et al. Science. Oct 25;254(5031):573-6 (1991). Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with 13C NMR. ] . In rats and mice, fasting promotes hepatic glucose production sustained by an increased pyruvate flux, and increases in PC activity and protein. Similar to other gluconeogenic enzymes, PC is positively regulated by glucagon and glucocorticoids while negatively regulated by insulin [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] .
Aside from the role of PC in gluconeogenesis, PC serves an anaplerotic role (an enzyme catalyzed reaction that can replenish the supply of intermediates in the citric acid cycle) for the tricarboxylic acid cycle (essential to provide oxaloacetate), when intermediates are removed for different biosynthetic purposes.
Role in Lipogenesis
In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor- g, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis [Meirleir, L.D. J of Child Neurology. Vol.17, Supplement 3 (2002). Defects of Pyruvate Metabolism and the Krebs Cycle. ] . PC is expressed at a very high level during the differentiation of adipocytes, and appears to be particularly important in adipose tissue, where it contributes to the generation of much of the NADPH required for lipogenesis. The generation of NADPH is coupled to the transport of mitochondrial acetyl groups into the cytosol for fatty acid synthesis. Acetyl-CoA is generated in the mitochondria by the oxidative decarboxylation of pyruvate, and, after condensation with oxaloacetate, acetyl groups are transported to the cytoplasm as citrate, which undergoes ATP-dependent cleavage to yield acetyl-CoA and oxaloacetate. This pathway requires a continuous supply of oxaloacetate, which is produced by PC. Acetyl-CoA, a building block for the synthesis of long-chain fatty acids, is then converted into malonyl-CoA by acetyl-CoA carboxylase (ACC). Meanwhile, the oxaloacetate generated in the cytosol from citrate is reduced with NADH to malate, which in turn is oxidatively decarboxylated in a reaction catalyzed by NADP+- dependent malate dehydrogenase. The pyruvate thereby produced is taken up by the mitochondria and carboxylated to give oxaloacetate, while the NADPH generated is used in the pathway of fatty acid synthesis [Reshef L, Hanson RW, Ballard FJ. J Biol Chem. Nov 25;245(22):5979-84 (1970). A possible physiological role for glyceroneogenesis in rat adipose tissue. ] .
PC is also involved in glyceroneogenesis, the process of synthesizing glycerol for esterification with free fatty acids to form triglycerides in differentiating adipocytes, as shown in Figure 3. The high rate of glyceroneogenesis is due to the presence of a high activity of the cytosolic PEPCK, which converts oxaloacetate to phosphoenolpyruvate (PEP). PEP is subsequently converted to glycerol via dihydroxyacetone phosphate for esterification with fatty acids to form triglycerides. As the cytosolic oxaloacetate is provided by PC through the citrate/pyruvate shuttle, PC is an important enzyme involved in glyceroneogenesis in differentiating adipocytes [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] [Reshef L, Hanson RW, Ballard FJ. J Biol Chem. Nov 25;245(22):5979-84 (1970). A possible physiological role for glyceroneogenesis in rat adipose tissue. ] .
The role of PC in the nervous system
Synapse junctions of neurons in both the central and peripheral nervous systems require neurotransmitters to transmit an electrical impulse. Glutamate is of particular interest as it is one of the excitatory neurotransmitters. Glutamate is synthesized in the nervous system by astrocytes (glial cells in the brain) before being converted to glutamine, which is secreted to the neuronal synapses. This cycle is known as the ‘glutamine-glutamate cycle’. The operation of the glutamine-glutamate cycle requires a continuous supply of oxaloacetate to be made available through the reaction catalyzed by PC, in order to provide σ-ketoglutarate. This is supported by the high activity of PC in astrocytes, where there are no activities of the other gluconeogenic enzymes present to perform gluconeogenesis. Pyruvate carboxylation appears to be a critical step in the pathway for synthesizing glutamate from TCA cycle intermediates. The importance of PC in the nervous system can be clearly seen in mental retardation in PC-deficient patients who carry one or more forms of mutations of the PC gene and by the brain abnormalities that severely compromise their psychomotor development [Gamberino WC, et al. J. Neurochem. 69: 2312–2325 (1997). Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.] .
The role of PC in pancreatic β-cells
In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the ‘pyruvate/malate cycle’ to shuttle 3C or 4C between mitochondria and cytoplasm [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] . Glucose is a potent stimulator of insulin secretion from β-pancreatic cells when extracellular levels are high (greater than 3 mM). This insulin secretion results in the rapid uptake of glucose by pancreatic β-cells. Signaling for glucose-induced insulin release is believed to require aerobic glycolysis plus TCA cycle activity. The activities of two mitochondrial enzymes, pyruvate dehydrogenase and PC, have been shown to be elevated when islets are grown in higher-than- physiological concentrations of glucose, suggesting that both enzymes are involved in the regulation of glucose-induced insulin release. In the islets the glucose undergoes oxidation to pyruvate, which is subsequently carboxylated by PC. A pyruvate-malate shuttle operates across the mitochondrial membrane, as shown in Figure 4. The high level of PC permits the rapid formation of oxaloacetate, which is subsequently converted into malate; this crosses the mitochondrial membrane to the cytosol, where it is decarboxylated to pyruvate by malate dehydrogenase, producing a putative coupling factor, NADPH [MacDonald MJ. J Biol Chem. Aug 25;270(34):20051-8 (1995). Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. ] .
Physiological States that Alter PC Expression
Different physiological conditions have been shown to alter the level of PC expression: these include nutritional alterations, diabetes, hormonal changes, neonatal development, adipogenesis and lactation [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] . Diabetes and hyperthyroidism increase the level of expression of pyruvate carboxylase in the long term, while its activity in the short term is controlled by the intramitochondrial concentrations of acetyl-CoA and pyruvate. Defects in the expression or biotinylation of pyruvate carboxylase in humans almost invariably results in early death or at best a severely debilitating psychomotor retardation, clearly reflecting the vital role it plays in intermediary metabolism in many tissues including the brain [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] .
Fasting in rats has been shown to induce 2±3-fold increases in hepatic PC activity. After refeeding a small increase in total PC activity has been detected in kidney. The hormonal mechanisms that regulate the total amount of PC activity during fasting and refeeding still remain unclear. Increases in the level of PC activity during starvation have been correlated with increases in the plasma concentrations of glucagon and glucocorticoids. PC activity has also been shown to be reduced by 50% in diabetes-prone rats fed a diet containing 6% menhaden oil, which is rich in long-chain highly unsaturated fatty acids [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
The rate of hepatic gluconeogenesis is increased dramatically in the diabetic state, concomitant with increases in the activities of all gluconeogenic enzymes, i.e. PEPCK, fructose-1,6-bisphosphatase, glucose-6-phosphatase and PC. In rats with induced diabetes, the hepatic PC activity was increased 2-fold over that of control rats. This increase in enzymic activity, which resulted from an increased amount of protein due to an enhanced rate of synthesis, is thought to be mediated by a high plasma glucagon-insulin ratio. Administration of insulin to diabetic rats brought the amount of PC and its activity back to the control levels [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
Apart from being both a gluconeogenic and a lipogenic enzyme, PC also plays an important role in glucose-induced insulin secretion in pancreatic islets, as described above. In the Goto-Kakizaki rat, a genetic model of type II (non-insulin dependent) diabetes in which glucose-induced insulin secretion in pancreatic b-cells is impaired, it has been found that PC activity was 45% of that in the normal rat islets, due to a decrease in the amount of PC protein. However, administration of insulin to Goto-Kakizaki rats resulted in the recovery of PC activity to that of normal rats [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
It has long been known that thyroid hormone affects the hepatic gluconeogenic rate in rats by increasing the activity of gluconeogenic enzymes, including PC. Experiments carried out by Weinberg and Utter showed that hepatic PC activity was increased 2-fold in hyperthyroid rats, whereas in hypothyroid rats PC was decreased 2-fold [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
Glucagon has long been demonstrated to increase the rate of pyruvate carboxylation in mitochondria isolated from rat hepatocytes, without changing the level of PC. Glucagon stimulates the respiratory chain, leading to an activation of pyruvate carboxylation. The increase in respiratory-chain activity (oxygen uptake) stimulates gluconeogenesis by generating ATP and by providing reducing equivalents to the cytosol. The increase in oxygen uptake therefore indirectly stimulates pyruvate uptake into the mitochondria [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
Adrenaline is also known to stimulate pyruvate carboxylation by isolated liver mitochondria. Little is known about the mechanism by which adrenaline acts on pyruvate metabolism, although it has been shown that adrenaline also acts via calcium- mediated pathways, similar to glucagon [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
As the maternal circulation provides glucose for the developing fetus, gluconeogenesis does not occur in fetal liver, but is triggered rapidly soon after birth. An increase in PC activity is accompanied by increases in the activities of other gluconeogenic enzymes, showing that the gluconeogenic pathway begins to function. The marked increase in PC activity during the suckling period has recently been shown to be concomitant with increases in PC protein and its transcripts [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
PC and genetic obesity
In genetically obese Zucker fatty rats, PC expression has been shown to be elevated 2±5-fold at the onset of obesity, along with an increased level of the liver/adipose-specific PC transcript C. This increase in PC levels is also accompanied by increases in the levels of other lipogenic enzymes, i.e. ACC, fatty acid synthase and ATP-citrate lyase. Given the lipogenic role of PC, as mentioned above, it has been proposed that oxaloacetate is consumed copiously in obesity, thus contributing to the hypertrophy of adipose tissue during the development of obesity [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
Given the diverse functions of PC described above, it is apparent that this enzyme plays very important roles in metabolism. This conclusion is supported by the effects of PC deficiency. Patients who suffer from the disease have less than 5%of normal PC activity. The main clinical features associated with a PC deficiency are congenital lactic acidosis and deterioration of the central nervous system. Lactic acidosis is associated with the deficit in both gluconeogenesis and TCA cycle activity, leading to an accumulation of alanine, lactate and pyruvate and a decrease in oxaloacetate and glucose [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
PC Deficiency is an autosomal recessive disorder, the gene for which is localized on chromosome 11q13.4-q13.5. Clinical presentation is highly variable, but there are 2 common presentations, each associated with a biochemical phenotype. The more severe form becomes symptomatic in the neonatal period, with increasing metabolic acidosis, ketosis, hepatomegaly, and death before 3 months of age [Meirleir, L.D. J of Child Neurology. Vol.17, Supplement 3 (2002). Defects of Pyruvate Metabolism and the Krebs Cycle] . These patients lack immunoreactive PC and its mRNA [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] . In the severe neonatal presentation, the lactate to pyruvate ratio is elevated, the 3-hydroxybutyric acid-to-acetoacetate ration is decreased, and postprandial hyperketonemia may be observed. Glutamine and aspartate levels are insufficient, resulting in elevation of citrulline and lysine levels. Impairment of the urea cycle leads to hyperammonemia [Meirleir, L.D. J of Child Neurology. Vol.17, Supplement 3 (2002). Defects of Pyruvate Metabolism and the Krebs Cycle] . In the neonatal presentation, lactic acidosis is associated with hypoglycemia, hyperammonemia, and ketosis with a high lactate to pyruvate ratio [Meirleir, L.D. J of Child Neurology. Vol.17, Supplement 3 (2002). Defects of Pyruvate Metabolism and the Krebs Cycle] .
A later-onset clinical presentation consists of delayed psychomotor development and chronic lactic acidemia with longer survival. Symptoms range from mild-to-moderate lactic acidaemia, delayed development and psychomotor retardation, but may survive for many years. These patients have some residual immunoreactive PC and mRNA [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] . The two forms of the disease have distinct ethnic groups in which they occur. The less severe form has been reported among North American native peoples, whereas the more severe form has been reported in the U.K. and France. The severity of PC deficiency may also be influenced by environmental factors, such as stress and fasting [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
Concluding Remarks and Future Directions
Early research on pyruvate carboxylase focused on the characterization of the physical properties and the kinetics of the enzyme. Since the development of recombinant DNA technology, information on the structure of the enzyme has been enormously enhanced by the cloning and sequencing of the genes and cDNA encoding this enzyme. The availability of various expression vectors will also facilitate the production of recombinant PC on a large scale for structure determination by X-ray crystallography. If the 3D structure of PC or its active site could be obtained by X-ray crystallography, it would greatly increase the probability of designing an inhibitor or agonist that could pharmacologically modulate PC activity [Jitrapakdeea et al. Cell. Mol. Life Sci. 63; 843–854 (2006). Anaplerotic roles of pyruvate carboxylase in mammalianTissues.] . This information should allow us to fully understand the relationship of structure to function for PC. The availability of cloned promoters of yeast and mammalian PC genes will also provide an excellent opportunity to investigate the role of regulatory proteins that mediate transcriptional regulation. In terms of clinical importance, PC deficiency has brought attention to the need for an understanding of the molecular biology of this defect in humans. The availability of embryonic stem cell technology should allow one to create a mouse model of PC deficiency by mimicking mutations that occur naturally in humans, or to investigate other physiological roles of PC by the gene knock-out approach [Wallace, J. C. and Jitrapakdee, S. Biochem. J. 340, 1-16 (1999). Structure, function and regulation of pyruvate carboxylase. ] .
A deficiency of pyruvate carboxylase can cause
lactic acidosisas a result of lactate build up. Normally, excess pyruvateis shunted into gluconeogenesisvia conversion of pyruvate into oxaloacetate, but because of the enzyme deficiency, excess pyruvate is converted into lactate instead. As a key role of gluconeogenesis is in the maintenance of blood sugar, deficiency of pyruvate carboxylase can also lead to hypoglycemia, among other things.
Pyruvate carboxylase deficiency
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