Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Full text links

Actions

Share

Review
.2008 Aug 1;413(3):369-87.
doi: 10.1042/BJ20080709.

Structure, mechanism and regulation of pyruvate carboxylase

Affiliations
Review

Structure, mechanism and regulation of pyruvate carboxylase

Sarawut Jitrapakdee et al. Biochem J..

Abstract

PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Anaplerotic role of PC in various mammalian tissues
Glucose is oxidized to pyruvate through the glycolytic pathway. PC replenishes oxaloacetate in the Krebs tricarboxylic acid (TCA) cycle when its intermediates are used for various biosynthetic pathways, depending on the tissues. In liver, oxaloacetate (OAA) is utilized as the precursor for gluconeogenesis whereby OAA can exit the mitochondria as malate before being converted back to OAA by cytosolic malate dehydrogenase (MDH). In some situations OAA is converted to phosphoenolpyruvate (PEP) by mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) [8,9]. Cytoplasmic OAA is converted to glucose by the combined activities of cytoplasmic PEPCK (PEPCK-C), fructose-1,6-bisphosphatase, glucose-6-phosphatase and 7 of the 10 glycolytic enzymes. In fat cells, PC provides oxaloacetate to facilitate the export of acetyl CoA in the form of citrate that leaves mitochondria forde novo fatty acid synthesis. Oxaloacetate liberated in the cytoplasm by citrate cleavage enzyme is converted to PEP by PEPCK-C as in the liver [8]. PEP is then converted to glycerol by a pathway known as “glyceroneogenesis”, while acetyl CoA is converted to malonyl-CoA by acetyl CoA carboxylase (ACC). Fatty acid synthase (FAS) catalyses the condensation of two-carbon moieties from malonyl-CoA to produce long chain acyl-CoA that are subsequently esterified with glycerol to form triglycerides. In pancreatic β-cells, PC is involved in a ‘pyruvate cycle’ that involves the exchange of TCA cycle intermediates and cytosolic pyruvate, catalysed by MDH and malic enzyme (ME) [30,31]. This cycle produces a large amount of NADPH which is one of the metabolic coupling factors. In astrocytes, α-ketoglutarate is converted by an aspartate aminotransferase (AAT) to glutamate, one of the neurotransmitter substances of neurons [39]. Key metabolites glyceraldehyde 3-phosphate (G-3-P), dihydroxyacetone phosphate (DHAP), and enzymes lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) are indicated.
Figure 2
Figure 2. Role of PC during metabolic adaptation
(A) In an over-nutrition situation, PC activity and pyruvate cycling activity are increased, resulting in increased insulin secretion in response to the chronically elevated level of plasma glucose [43]. Hepatic glucose production is still properly maintained in this condition. Adipocyte mass is expanded concomitant with increased expression of PC and other enzymes of fat synthesis. (B) In obesity-induced type 2 diabetes, chronic exposure of β-cells to an elevated level of glucose due to peripheral insulin resistance impairs β-cell function. Excessive hepatic glucose production results in an elevated level of plasma glucose, creating a “glucotoxicity loop”. Severe hyperglycemia reduces expression of several β-cell specific genes including PC. Mitochondrial metabolism of adipocytes is also impaired, causing down-regulation of some lipogenic enzymes including PC. Triglycerides (TG) and free fatty acids (FFA) released from adipose tissue to plasma further impair β-cell function and PC gene expression, creating a “lipotoxicity loop”. [54]
Figure 3
Figure 3. Tissue-specific regulation of PC in mammals
The PC gene is regulated by two alternative promoters, the proximal (P1) and the distal (P2). In adipose tissue and gluconeogenic tissue (liver), the P1 promoter is active and is responsible for the production of PC mRNA that contains identical 5’-untranslated region exons [77, 78]. In adipocytes, the P1 promoter is regulated by peroxisome proliferator-activated receptor gamma (PPARγ) [23], while in liver it is still unclear whether PPARγ alone or other factors, including hepatocyte nuclear factor 4α (HNF4α) and cAMP-responsive element binding protein (CREB), may also contribute to regulation in this tissue [77, 78, 92]. In pancreatic β-cells PC is regulated by the P2 promoter with an interplay of basal [specific protein (Sp1/Sp3) and upstream stimulatory factors (USFs)] and β-cell specific transcription factors [84, 85]. It is not known whether v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (Mafa1) and pancreatic-duodenal homeobox 1 (Pdx 1) directly regulate P2 activity.
Figure 4
Figure 4. Domain architecture of PC
(A) Schematic drawing of the primary structure arrangement for the multidomain PC. The central allosteric domain is indicated with a star [103]. (B) The structure of theStaphylococcus aureus PC monomer B [61]. The BC, CT, BCCP and allosteric domains are coloured blue, yellow, red and green, respectively. The chemical reactions catalysed in the individual domains are illustrated below the corresponding domain structure.
Figure 5
Figure 5. The acetyl CoA binding site inR. etli PC [103]
(A) The nucleotide portion of ethyl-CoA (shown as spheres), a non-hydrolysable analogue of acetyl CoA, is bound at the N-terminal end of the allosteric domain’s central spanning helix and near the dimerization interface for the BC domain. The domains are coloured in dark blue (BC domain 1), light blue (BC domain 2), green (allosteric domain) and yellow (CT domain). (B) Interactions with the nucleotide portion of ethyl-CoA include residues from both BC subunits and from the allosteric domain. Reproduced from [103].
Figure 6
Figure 6. The quaternary structure ofR. etli PC
(A) Schematic representation of the arrangement of the individual monomers making up the tetramers. The arrows represent the general N-terminal to C-terminal direction of the individual polypeptide chains. The arrangement of the tetramer yields two distinct faces, with the monomers running antiparallel on each face and perpendicularly between the faces. (B) Model of the PC tetramer showing the movement of the BCCP domain between neighbouring active sites on opposing polypeptide chains [103]. (C) Surface representation of the top face of the tetramer with one of the two monomers outlined in black for clarity. The location of the ligand binding sites and the distances between them are indicated [103]. (D) Surface representation of the bottom face of the tetramer after a 180° rotation about the y axis. The distance between opposing active sites increases to 80 Å as a result of the altered orientation of the BC domain. The BCCP domain is disordered on the bottom face of the PC tetramer fromR. etli and, consequently, is not modelled into the structure [103].A,B andC are reproduced from [103].
Figure 7
Figure 7. The exo biotin binding pocket in bacterial PC
(A) Cartoon representation of the bottom face of SaPC, where biotin rests in the exo binding site (circled) of theopposing polypeptide chain. One monomer is coloured in blue, green, yellow and red to indicate the BC, allosteric, CT and BCCP domains, respectively. The second monomer is coloured in light blue. The exo binding site is located on the C-terminal end of the allosteric domain’s central spanning helix and at the interface with the CT domain. (B) Biotin in the exo binding site in RePC. The Fo - Fc electron density omit map for biotinylated Lys1114 in RePC was contoured at 2.5σ. (c) Overlay of the allosteric domain in monomer B of SaPC (in colours) and the allosteric domain from the bottom face monomer of RePC (grey). The exo binding pockets are very similar between the two species and each site provides similar contacts with the biotin moiety. However, the binding position of biotin in the exo binding site is completely inverted between RePC and SaPC. The BCCP domain in RePC is disordered, as indicated by the dashed grey line.
Figure 8
Figure 8
(A) Chemical mechanism for the carboxylation of biotin via a carboxyphosphate intermediate.(B) Chemical mechanism for the carboxylation of pyruvate by carboxybiotin. B represents a basic residue in the enzyme active site.
Figure 9
Figure 9
The active site of 5S showing the bound cobalt ion (pink sphere), its water ligands, and side chains of residues which interact with either the cobalt ion or substrate (ball and stick) are shown. Potential interactions involving the metal ion and active site ligands are represented by pink and grey dashed lines, respectively.(A) Free 5S, showing carbamylated lysine (K184) coordinating the cobalt ion.(B) 5S–oxaloacetate complex, showing the noncarbamylated K184 interacting with cysteine-154 (C154) and oxaloacetate interacting with the cobalt ion.(C) 5S-pyruvate complex and two conformations of carbamylated/noncarbamylated K184 (both refined at half occupancy). WATA and WATB are water molecules. Adapted with permission from Macmillan Publishers Ltd: (EMBO Journal) [132], copyright (2004).
Figure 10
Figure 10
Part of a sequence alignment (ClustalW) showing the sequences of pyruvate carboxylase fromB. thermodenitrificans (BTHERM)[146],R. etli (RHI)[103],S. aureus (STA)[61] and humans (HUM)[61] and that of the transcarboxylase 5S subunit fromP. shermanii (5S)[109]. Identical residues are outlined in grey residues. Asterisks indicate conserved residues for which functions are described in Section 8.3. Single dots indicate every 10th residue.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Utter MF, Keech DB. Formation of oxaloacetate from pyruvate and carbon dioxide. J. Biol. Chem. 1960;235:17–18. - PubMed
    1. Owen OE, Kalhan SC, Hanson RW. The key role of anaplerosis and cataplerosis for citric acid cycle function. J. Biol. Chem. 2002;277:30409–30412. - PubMed
    1. Jitrapakdee S, Wallace JC. Structure, function and regulation of pyruvate carboxylase. Biochem J. 1999;340:1–16. - PMC - PubMed
    1. Sauer U, Eikmanns BJ. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 2005;29:765–794. - PubMed
    1. de Jong-Gubbels P, Bauer J, Niederberger P, Stückrath I, Kötter P, van Dijken JP, Pronk JT. Physiological characterisation of a pyruvate-carboxylase- negative Saccharomyces cerevisiae mutant in batch and chemostat cultures. Antonie Van Leeuwenhoek. 1998;74:253–263. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp