View source for Pyruvate carboxylase

{{enzyme
| Name = Pyruvate carboxylase
| EC_number = 6.4.1.1
| CAS_number = 9014-19-1
| IUBMB_EC_number = 6/4/1/1
| GO_code = 0004736
| image = Pyruvate_Carboxylase_fromPDB_2QF7.png
| width =
| caption = [[X-ray crystallography#Biological macromolecular crystallography|Crystallographic structure]] of pyruvate carboxylase from ''[[Rhizobium|Rhizobium etli]]'': biotin carboxylase domain (blue); allosteric linking domain (green); biotin binding domain (red); and carboxyl transferase domain (orange)<ref name="pmid18613815">{{PDB|2QF7}}; {{cite journal |vauthors=Jitrapakdee S, St Maurice M, Rayment I, Cleland WW, Wallace JC, Attwood PV | title = Structure, mechanism and regulation of pyruvate carboxylase | journal = Biochem. J. | volume = 413 | issue = 3 | pages = 369–87 |date=August 2008 | pmid = 18613815 | pmc = 2859305 | doi = 10.1042/BJ20080709 | url = | issn = }}</ref>
}}
{{Pfam box |Symbol = PYR_CT 
| Name = Pyruvate carboxyltransferase 
| Pfam = PF00682 
| InterPro = IPR000891 
| PROSITE = PDOC50991 
}}
{{Infobox protein
   |Name=Pyruvate carboxylase
   |image=
   |caption=
   |Symbol=PC
   |AltSymbols=
   |HGNCid=8636
   |Chromosome=11
   |Arm=q
   |Band=11
   |LocusSupplementaryData=-q13.1
   |ECnumber=6.4.1.1
   |OMIM=608786
   |EntrezGene=5091
   |RefSeq=NM_000920
   |UniProt=P11498
   |PDB=
}}

{{Infobox_gene}}

'''Pyruvate carboxylase''' ('''PC''') encoded by the gene PC is an [[enzyme]] of the [[ligase]] class that [[catalyzes]] (depending on the species) the physiologically irreversible [[carboxylation]] of [[pyruvate]] to form [[oxaloacetate]] (OAA).

<gallery>
 Image:Pyruvic-acid-2D-skeletal.png |[[Pyruvic acid]]
 Image:Oxaloacetic acid.png |[[Oxaloacetic acid]]
</gallery>

The reaction it catalyzes is:
:pyruvate + {{chem|H|C|O|3|-}} + ATP → oxaloacetate + ADP + P

It is an important [[Anaplerotic reactions|anaplerotic reaction]] that creates oxaloacetate from pyruvate. The enzyme is a mitochondrial [[protein]] containing a [[biotin]] prosthetic group,<ref name="pmid18613815"/> requiring [[magnesium]] or [[manganese]] and acetyl CoA.

Pyruvate carboxylase was first discovered in 1959 at [[Case Western Reserve University]] by M. F. Utter and D. B. Keech.<ref name="pmid13840551">{{cite journal |vauthors=Utter MF, Keech DB | title = Formation of oxaloacetate from pyruvate and carbon dioxide | journal = J. Biol. Chem. | volume = 235 | issue = | pages = PC17–8 |date=May 1960 | pmid = 13840551 | doi = | url = | issn = }}</ref><ref name="pmid762171">{{cite journal |vauthors=Cohen ND, Beegen H, Utter MF, Wrigley NG | title = A re-examination of the electron microscopic appearance of pyruvate carboxylase from chicken liver | journal = J. Biol. Chem. | volume = 254 | issue = 5 | pages = 1740–7 |date=March 1979 | pmid = 762171 | doi = | url = http://www.jbc.org/cgi/pmidlookup?view=long&pmid=762171 | issn = }}</ref> Since then it has been found in a wide variety of prokaryotes and eukaryotes including fungi, bacteria, plants, and animals.<ref name="pmid16505973">{{cite journal |vauthors=Jitrapakdee S, Vidal-Puig A, Wallace JC | title = Anaplerotic roles of pyruvate carboxylase in mammalian tissues | journal = Cell. Mol. Life Sci. | volume = 63 | issue = 7–8 | pages = 843–54 |date=April 2006 | pmid = 16505973 | doi = 10.1007/s00018-005-5410-y | url = }}</ref> In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of [[neurotransmitter]]s, and in glucose-induced insulin secretion by pancreatic islets. Oxaloacetate produced by PC is an important intermediate, which is used in these biosynthetic pathways.<ref name="pmid12150961">{{cite journal |vauthors=Jitrapakdee S, Nezic MG, Cassady AI, Khew-Goodall Y, Wallace JC | title = Molecular cloning and domain structure of chicken pyruvate carboxylase | journal = Biochem. Biophys. Res. Commun. | volume = 295 | issue = 2 | pages = 387–93 |date=July 2002 | pmid = 12150961 | doi = 10.1016/S0006-291X(02)00651-4 | url = }}</ref> 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.<ref name="pmid8687459">{{cite journal |vauthors=Jitrapakdee S, Walker ME, Wallace JC | title = Identification of novel alternatively spliced pyruvate carboxylase mRNAs with divergent 5'-untranslated regions which are expressed in a tissue-specific manner | journal = Biochem. Biophys. Res. Commun. | volume = 223 | issue = 3 | pages = 695–700 |date=June 1996 | pmid = 8687459 | doi = 10.1006/bbrc.1996.0958 | url = }}</ref>

==Structure==

Structural studies of PC have been conducted by [[electron microscopy]], by limited [[proteolysis]], and by cloning and gasa 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 transport [[carbon dioxide]] to the catalytic site that is formed at the interface between adjacent monomers. Each subunit of the functional tetramer contains four domains: the biotin carboxylation (BC) domain, the transcarboxylation (CT) domain,  the biotin carboxyl carrier (BCCP) domain and the recently termed PC tetramerization (PT) domain.<ref name="pmid14993673">{{cite journal |vauthors=Kondo S, Nakajima Y, Sugio S, Yong-Biao J, Sueda S, Kondo H | title = Structure of the biotin carboxylase subunit of pyruvate carboxylase from Aquifex aeolicus at 2.2 A resolution | journal = Acta Crystallogr. D | volume = 60 | issue = Pt 3 | pages = 486–92 |date=March 2004 | pmid = 14993673 | doi = 10.1107/S0907444904000423 | url = }}</ref><ref name="pmid19523900">{{cite journal |vauthors=Yu LP, Xiang S, Lasso G, Gil D, Valle M, Tong L | title = A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A | journal = Structure | volume = 17 | issue = 6 | pages = 823–32 |date=June 2009 | pmid = 19523900 | doi = 10.1016/j.str.2009.04.008 | url = | issn = | pmc = 2731552 }}</ref> From the two most complete crystal structures available, an asymmetric and symmetric form of the protein have been visualized.<ref name="pmid17717183">{{cite journal |vauthors=St Maurice M, Reinhardt L, Surinya KH, Attwood PV, Wallace JC, Cleland WW, Rayment I | title = Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme | journal = Science | volume = 317 | issue = 5841 | pages = 1076–9 |date=August 2007 | pmid = 17717183 | doi = 10.1126/science.1144504 | url = }}</ref> The ''[[Staphylococcus aureus]]'' tetramer in complex with the activator coenzyme A is highly symmetric, possessing 222 symmetry, and has been confirmed by cryo-EM studies.<ref name="pmid19523900"/>  In contrast the ''[[Rhizobium|Rhizobium etli]]'', tetramer in complex with ethyl-CoA, a non-hydrolyzable analog of [[acetyl-CoA]], possesses only one line of symmetry.<ref name="pmid17717183"/>

{{multiple image|align=center
| header  = Pyruvate Carboxylase Symmetry Comparison
| width  = 200
| height = 150
| lines  = 2
|image1=Pyruvate Carboxylase 2QF7, sswilson7.png|caption1=''[[Rhizobium|Rhizobium etli]]''
|image2=Pyruvate carboxylase 3HO8, sswilson7.png|caption2=''[[Staphylococcus aureus]]''
}}

Pyruvate carboxylase uses a covalently attached [[biotin]] cofactor which is used to catalyze the [[Adenosine triphosphate|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.<ref name="pmid17717183"/>

==Reaction mechanism==

[[File:Mechanism of Pyruvate Carboxylase, 5-15-2010, sswilson7.png|thumb|right | 300px | alt = black & white schematic diagram depicting the mechanism of pyruvate carboxylase | Proposed mechanism of pyruvate carboxylase:<br />('''A''') ATP dependent carboxylation of biotin (BC domain);<br />('''B''') Transcarboxylation of pyruvate (CT domain).]]

The reaction mechanism can be subdivided into two partial reactions (see figure to the right). In the first reaction, [[adenosine triphosphate|ATP]] is carboxylated to produce carbonic phosphoric anhydride [<sup>−</sup>O(<sup>−</sup>O)P(=O)O–C(=O)O<sup>−</sup>] which in turn carboxylates a [[biotin]] cofactor that is covalently attached to a lysine residue of the BCCP domain.<ref name="pmid16505973"/>  Carbonic phosphoric anhydride decomposes into carbon dioxide and phosphate prior to attack by the enzyme linked biotin molecule.  In most species, this reaction requires [[acetyl-CoA]] as an allosteric activator binding to the PT domain.<ref name="pmid19523900"/> In the second reaction, occurring in the CT domain of an adjacent monomer, carbon dioxide is transferred to the acceptor molecule, pyruvate, to form oxaloacetate. The reaction proceeds via the removal of a proton from pyruvate, by an as yet unidentified active site residue, to generate an [[enol]]ate intermediate.  The enolate intermediate then attacks CO<sub>2</sub> transiently released from the enzyme linked biotin molecule.  The resultant [[oxaloacetate]] is released.  The biotin molecule is protonated by the aforementioned active site residue and released from the active site of the CT domain to be recarboxylated.<ref name="pmid19523900"/><ref name="pmid17717183"/> 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.<ref name="pmid12150961"/>

==Function==

During [[gluconeogenesis]], pyruvate carboxylase is involved in the synthesis of [[phosphoenolpyruvate]] (PEP) from [[pyruvate]].  Pyruvate is first converted by pyruvate carboxylase to [[oxaloacetate]] (OAA) in the mitochondrion requiring [[hydrolysis]] of one molecule of [[Adenosine triphosphate|ATP]]. The OAA is then decarboxylated and simultaneously phosphorylated, which is catalyzed by one of two isoforms of [[phosphoenolpyruvate carboxykinase]] (PEPCK) either in the [[cytosol]] or in the [[mitochondria]] to produce PEP. Under ordinary gluconeogenic conditions, OAA is converted into PEP by mitochondrial PEPCK; the resultant PEP is then transported out of the mitochondrial matrix by an anion transporter carrier system,<ref name="pmid19635791">{{cite journal  |vauthors=Stark R, Pasquel F, Turcu A, etal | title =  Phosphoenolpyruvate cycling via mitochondrial phosphoenolpyruvate carboxykinase links anaplerosis and mitochondrial GTP with insulin secretion. | journal = Journal of Biological Chemistry | volume = 284 | pages = 26578–26590 | year = 2009 | pmid=19635791 | issue = 39 | doi=10.1074/jbc.M109.011775 | pmc=2785346}}</ref> and converted into glucose by cytosolic gluconeogenic enzymes.  However, during starvation when cytosolic NADH concentration is low and mitochrondrial NADH levels are high oxaloacetate can be used as a shuttle of reducing equivalents.  As such OAA is converted into [[malic acid|malate]] by mitochondrial [[Malate dehydrogenase]] (MDH).  After export into the cytosol, malate is converted back into OAA, with concomitant reduction of NAD<sup>+</sup>; OAA is subsequently converted to PEP which is available for gluconeogenesis in the cytosol along with the transported reducing equivalent NADH.<ref name="pmid18613815"/>

Very high levels of PC activity, together with high activities of other gluconeogenic enzymes including [[PEPCK]], [[fructose-1,6-bisphosphatase]] and [[glucose-6-phosphatase]] in liver and kidney cortex, suggest that a primary role of PC is to participate in gluconeogenesis in these organs. 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 is elevated.<ref name="pmid1948033">{{cite journal |vauthors=Rothman DL, Magnusson I, Katz LD, Shulman RG, Shulman GI | title = Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with 13C NMR | journal = Science | volume = 254 | issue = 5031 | pages = 573–6 |date=October 1991 | pmid = 1948033 | doi = 10.1126/science.1948033 }}</ref> In rats and mice, alteration of nutrition status has been shown to affect hepatic PC activity.<ref name="pmid11448844">{{ cite journal |vauthors=Bizeau ME, Short C, Thresher JS, Commerford SR, Willis WT, Pagliassotti MJ | title = Increased pyruvate flux capacities account for diet induced increase in gluconeogenesis ''in vitro'' | journal = Am. J. Physiol. Regul. Integr. Comp. Physiol. | volume = 281 | issue =2 | pages = R427–R433| year = 2001 | pmid = 11448844}}</ref> Fasting promotes hepatic glucose production sustained by an increased pyruvate flux, and increases in PC activity and protein concentration; diabetes similarly increases gluconeogenesis through enhanced uptake of substrate and increased flux through liver PC in mice and rats.<ref name="pmid9127680" >{{ cite journal |vauthors=Salto R, Sola M, Olicer FJ, Vargas AM | title = Effects of starvation, diabetes, and carbon tetrachloride intoxication on rat kidney cortex and liver pyruvate carboxylase levels | journal = Arch. Physiol. Biochem. | volume = 104 | issue = 7 |pages = 845–850 |date=Dec 1996 | pmid = 9127680 | doi = 10.1076/apab.104.7.845.13111 }}</ref><ref name="pmid10342812">{{ cite journal | doi = 10.2337/diabetes.48.6.1251 |vauthors=Large V, Beylot M | title = Modifications of citric acid cycle activity and gluconeogenesis in strepozotocin induced diabetes and effects of metformin | journal = Diabetes | volume = 48 | issue = 6 | pages = 1251–1257 |date=June 1999 | pmid = 10342812 }}</ref> Similarly to other gluconeogenic enzymes, PC is positively regulated by [[glucagon]] and [[glucocorticoid]]s while negatively regulated by [[insulin]].<ref name="pmid16505973" />  Further supporting the key role of PC in gluconeogenesis, in dairy cattle, which have [[hexose]] absorption ability at adequate nutrition levels, PC and the associated gluconeogenic enzyme PEPCK are markedly elevated during the transition to lactation in proposed support of lactose synthesis for milk production.<ref name ="pmid10877388">{{ cite journal |vauthors=Greenfield RB, Cecava MJ, Donkin SS | title = Changes in mRNA expression for gluconeogenic enzymes in the liver of dairy cattle during transition to lactation | journal = [[Journal of Dairy Science]] | volume =  83| issue = 6 | pages = 1228–1236 | year = 2002 | pmid = 10877388 | doi = 10.3168/jds.S0022-0302(00)74989-7 }}</ref>

Aside from the role of PC in gluconeogenesis, PC serves an [[anaplerotic reactions|anaplerotic]] role (an enzyme catalyzed reaction that can replenish the supply of intermediates in the citric acid cycle) for the [[citric acid cycle|tricarboxylic acid cycle]] (essential to provide oxaloacetate), when intermediates are removed for different biosynthetic purposes.

{{GlycolysisGluconeogenesis_WP534|highlight=Pyruvate_carboxylase}}

==Clinical significance==

As a crossroad between [[carbohydrate metabolism|carbohydrate]] and [[lipid metabolism|lipid]] metabolism, pyruvate carboxylase expression in gluconeogenic tissues, adipose tissues and pancreatic islets must be coordinated.  In conditions of over nutrition, PC levels are increased in pancreatic  β-cells to increase [[pyruvate cycling]] in response to chronically elevated levels of [[glucose]].<ref name="pmid15507531">{{ cite journal |vauthors=Liu YQ, Han J, Epstein PN, Long YS |title= Enhanced rat  β-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway | journal =  Am. J. Physiol. Endocrinol. Metab. | volume = 288 | issue = 3 |pages = E471–E478 |date=Dec 2005 | pmid = 15507531 | doi =10.1152/ajpendo.00427.2004 }}</ref>  In contrast, PC enzyme levels in the liver are decreased by [[insulin]];<ref name="pmid16601267">{{cite journal |vauthors=Desvergne B, Michalik L, Wahli W | title =  Transcriptional regulation of metabolism | journal = Physiol. Rev. | volume = 86 | issue = 2 | pages= 465–514 |date=April 2006 | pmid =16601267 |doi = 10.1152/physrev.00025.2005 }}</ref> during periods of overnutrition adipocyte tissue is expanded with extreme expression of PC and other lipogenic enzymes.<ref name="pmid8687459"/><ref name= "pmid1375435">{{ cite journal |vauthors=Lynch CJ, McCall KM, Billingsley ML, Bohlen LM, Hreniuk SP, Martin LF, Witters LA, Vannucci SJ | title = Pyruvate carboxylase in genetic obesity | journal = Am. J. Physiol. | volume = 262 | pages = E608–E618 |date=May 1992 | pmid = 1375435 | issue = 5 Pt 1 }}</ref> Hepatic control of glucose levels is still regulated in an over nutrition situation, but in obesity induced type 2 diabetes the regulation of peripheral glucose levels is no longer under regulation of insulin.
In [[diabetes mellitus type 2|type 2 diabetic]] rats, chronic exposure of [[beta cell|β-cells]] to glucose due to peripheral [[insulin]] resistance results in decreased PC enzyme activity and decreased [[pyruvate]] cycling<ref name ="pmid8866570" >{{ cite journal | doi = 10.2337/diabetes.45.11.1626 |vauthors=MacDonald MJ, Tang J, Polonsky KS | title = Low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic islets of Zucker diabetic fatty rats | journal =Diabetes | volume = 45 | issue = 11 | pages = 1626–1630 |date=Nov 1996 | pmid = 8866570 }}</ref><ref name="pmid8666138" >{{ cite journal | doi = 10.2337/diabetes.45.7.886 |vauthors=McDonald MJ, Efendic S, Ostenson CG | title = Normalization by insulin of low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic islets of the GK rat | journal = Diabetes | volume = 45 | issue =7 |date=July 1996 | pmid = 8666138 | pages = 886–890 }}</ref> The continued overproduction of glucose by [[hepatocytes]] causes dramatic alteration of [[beta-cell|β-cell]] [[gene]] expression with large increases in normally suppressed genes, and  equivalent decreases in expression of mRNA for insulin, ion pumps necessary for insulin secretion, and metabolic enzymes related to insulin secretion, including pyruvate carboxylase<ref name="pmid1243814">{{ cite journal |vauthors=Laybutt DR, Glandt M, Xu G, Ahn YB, Trivedi N, Bonner-Weir S, Weir GC | title = Critical reduction in β-cell mass results in two distinct outcomes over time. Adaption with impaired glucose tolerance or decompensated diabetes | journal =J. Biol. Chem. | volume = 278 | issue = 5 | pages = 2997–3005 |date=Jan 2003 | pmid = 12438314 | doi = 10.1074/jbc.M210581200 }}</ref><ref name="pmid11796484">{{ cite journal | doi = 10.1210/en.143.2.339 |vauthors=Poitout V, Robertson RP | title = Secondary ß-cell failure in type 2 diabetes-a convergence of glucotoxicity and lipotoxicity | journal = Endocrinology | volume = 143 | issue = 2 | pages = 339–342 |date=Feb 2002 | pmid =11796484 }}</ref> Concurrently adipose tissue develops insulin resistance causing accumulation of triaglycerols and non-esterified [[fatty acids]] in circulation; these not only further impairing β-cell function,<ref name ="pmid11796484"/><ref name ="pmid 15073188">{{ cite journal |vauthors=Boucher A, Lu D, Burgess SC, Telamaque-Potts S, Jensen MV, Mulder H, Wang MY, Unger RH, Sherry AD, Newgard CB | title = Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue | journal = J. Biol. Chem. | volume = 279| issue = 26 | pages = 27263–27271 | year = 2004 |pmid = 15073188 | doi = 10.1074/jbc.M401167200 }}</ref> but also further decreasing PC expression.<ref name= "pmid11916915" >{{ cite journal | doi = 10.2337/diabetes.51.4.977 |vauthors=Busch AK, Cordery D, Denyer GS, Biden TJ | title= Expression profiling of palmitate- and oleate-regulated genes provides novel insights into the effects of chronic exposure on pancreatic β-cell function | journal = Diabetes | volume = 51| issue = 4| pages = 977–987 |date=Apr 2002 | pmid = 11916915 }}</ref><ref name ="pmid11781146">{{ cite journal |vauthors=Iizuka K, Nakajima H, Namba M, Miyagawa J, Mijazaki J, Hanafusa T, Matsuzawa Y |  title= Metabolic consequences of long-term exposure of pancreatic β-cells to free fatty acid with special reference to glucose insensitivity | journal = Biochim. Biophys. Acta | volume = 1586 | issue = 1| pages = 23–31|date=Jan 2002 | pmid = 11781146 | doi=10.1016/s0925-4439(01)00082-5}}</ref> These changes result in the decline of the [[beta-cell|β-cell]] phenotype in decompensated diabetes.

A [[Pyruvate carboxylase deficiency|deficiency of pyruvate carboxylase]] can cause [[lactic acidosis]] as a result of [[lactic acid|lactate]] build up.<ref name="pmid16278852">{{cite journal |vauthors=García-Cazorla A, Rabier D, Touati G, Chadefaux-Vekemans B, Marsac C, de Lonlay P, Saudubray JM | title = Pyruvate carboxylase deficiency: metabolic characteristics and new neurological aspects | journal = Ann. Neurol. | volume = 59 | issue = 1 | pages = 121–7 |date=January 2006 | pmid = 16278852 | doi = 10.1002/ana.20709 | url = | issn = }}</ref> Normally, excess [[pyruvate]] is shunted into [[gluconeogenesis]] via conversion of pyruvate into [[oxaloacetate]], but because of the enzyme deficiency, excess pyruvate is converted into [[lactic acid|lactate]] instead. As a key role of gluconeogenesis is in the maintenance of [[blood sugar]], deficiency of pyruvate carboxylase can also lead to [[hypoglycemia]].

==See also==
* [[Pyruvate carboxylase deficiency]]

==References==
{{Reflist|30em}}

==External links==
* [https://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=pdc  GeneReviews/NCBI/NIH/UW entry on Pyruvate Carboxylase Deficiency]

{{Glycolysis enzymes}}
{{Citric acid cycle enzymes}}
{{metabolism|state=uncollapsed}}
{{Carbon-carbon ligases}}
{{Enzymes}}
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{{DEFAULTSORT:Pyruvate Carboxylase}}
[[Category:EC 6.4.1]]
[[Category:Moonlighting proteins]]