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Phosphoenolpyruvate carboxykinase
Phosphoenolpyruvate carboxykinase
Phosphoenolpyruvate carboxykinase
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Phosphoenolpyruvate carboxykinase
PDB rendering based on 1khb.
Identifiers
SymbolPEPCK
PfamPF00821
InterProIPR008209
PROSITEPDOC00421
SCOP21khf / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1khb​, 1khe​, 1khf​, 1khg​, 1m51​, 1nhx​, 2gmv​
phosphoenolpyruvate carboxykinase 1 (soluble)
Phosphoenolpyruvate carboxykinase (GTP, cytosolic) monomer, Human
Identifiers
SymbolPCK1
Alt. symbolsPEPCK-C
NCBI gene5105
HGNC8724
OMIM261680
RefSeqNM_002591
Other data
EC number4.1.1.32
LocusChr. 20 q13.31
phosphoenolpyruvate carboxykinase 2 (mitochondrial)
Identifiers
SymbolPCK2
Alt. symbolsPEPCK-M, PEPCK2
NCBI gene5106
HGNC8725
OMIM261650
RefSeqNM_001018073
Other data
EC number4.1.1.32
LocusChr. 14 q12

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32, PEPCK) is an enzyme in the lyase family used in the metabolic pathway of gluconeogenesis. It converts oxaloacetate into phosphoenolpyruvate and carbon dioxide.[1][2][3]

It is found in two forms, cytosolic and mitochondrial.

Structure

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In humans there are two isoforms of PEPCK; a cytosolic form (SwissProt P35558) and a mitochondrial isoform (SwissProt Q16822) which have 63.4% sequence identity. The cytosolic form is important in gluconeogenesis. However, there is a known transport mechanism to move PEP from the mitochondria to the cytosol, using specific membrane transport proteins.[4][5][6][7][8] PEP transport across the inner mitochondrial membrane involves the mitochondrial tricarboxylate transport protein and to a lesser extent the adenine nucleotide carrier. The possibility of a PEP/pyruvate transporter has also been put forward.[9]

X-ray structures of PEPCK provide insight into the structure and the mechanism of PEPCK enzymatic activity. The mitochondrial isoform of chicken liver PEPCK complexed with Mn2+, Mn2+-phosphoenolpyruvate (PEP), and Mn2+-GDP provides information about its structure and how this enzyme catalyzes reactions.[10] Delbaere et al. (2004) resolved PEPCK in E. coli and found the active site sitting between a C-terminal domain and an N-terminal domain. The active site was observed to be closed upon rotation of these domains.[11]

Phosphoryl groups are transferred during PEPCK action, which is likely facilitated by the eclipsed conformation of the phosphoryl groups when ATP is bound to PEPCK.[11]

Since the eclipsed formation is one that is high in energy, phosphoryl group transfer has a decreased energy of activation, meaning that the groups will transfer more readily. This transfer likely happens via a mechanism similar to SN2 displacement.[11]

In different species

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PEPCK gene transcription occurs in many species, and the amino acid sequence of PEPCK is distinct for each species.

For example, its structure and its specificity differ in humans, Escherichia coli (E. coli), and the parasiteTrypanosoma cruzi.[12]

Mechanism

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PEPCKase converts oxaloacetate into phosphoenolpyruvate and carbon dioxide.

As PEPCK acts at the junction between glycolysis and the Krebs cycle, it causes decarboxylation of a C4 molecule, creating a C3 molecule. As the first committed step in gluconeogenesis, PEPCK decarboxylates and phosphorylates oxaloacetate (OAA) for its conversion to PEP, when GTP is present. As a phosphate is transferred, the reaction results in a GDP molecule.[10] When pyruvate kinase – the enzyme that normally catalyzes the reaction that converts PEP to pyruvate – is knocked out in mutants of Bacillus subtilis, PEPCK participates in one of the replacement anaplerotic reactions, working in the reverse direction of its normal function, converting PEP to OAA.[13] Although this reaction is possible, the kinetics are so unfavorable that the mutants grow at a very slow pace or do not grow at all.[13]

Function

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Gluconeogenesis

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PEPCK-C catalyzes an irreversible step of gluconeogenesis, the process whereby glucose is synthesized. The enzyme has therefore been thought to be essential in glucose homeostasis, as evidenced by laboratory mice that contracted diabetes mellitus type 2 as a result of the overexpression of PEPCK-C.[14]

The role that PEPCK-C plays in gluconeogenesis may be mediated by the citric acid cycle, the activity of which was found to be directly related to PEPCK-C abundance.[15]

PEPCK-C levels alone were not highly correlated with gluconeogenesis in the mouse liver, as previous studies have suggested.[15] While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). PEPCK-M has gluconeogenic potential per se.[2] Therefore, the role of PEPCK-C and PEPCK-M in gluconeogenesis may be more complex and involve more factors than was previously believed.

Animals

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In animals, this is a rate-controlling step of gluconeogenesis, the process by which cells synthesize glucose from metabolic precursors. The blood glucose level is maintained within well-defined limits in part due to precise regulation of PEPCK gene expression. To emphasize the importance of PEPCK in glucose homeostasis, over expression of this enzyme in mice results in symptoms of type II diabetes mellitus, by far the most common form of diabetes in humans. Due to the importance of blood glucose homeostasis, a number of hormones regulate a set of genes (including PEPCK) in the liver that modulate the rate of glucose synthesis.

PEPCK-C is controlled by two different hormonal mechanisms. PEPCK-C activity is increased upon the secretion of both cortisol from the adrenal cortex and glucagon from the alpha cells of the pancreas. Glucagon indirectly elevates the expression of PEPCK-C by increasing the levels of cAMP (via activation of adenylyl cyclase) in the liver which consequently leads to the phosphorylation of S133 on a beta sheet in the CREB protein. CREB then binds upstream of the PEPCK-C gene at CRE (cAMP response element) and induces PEPCK-C transcription. Cortisol on the other hand, when released by the adrenal cortex, passes through the lipid membrane of liver cells (due to its hydrophobic nature it can pass directly through cell membranes) and then binds to a Glucocorticoid Receptor (GR). This receptor dimerizes and the cortisol/GR complex passes into the nucleus where it then binds to the Glucocorticoid Response Element (GRE) region in a similar manner to CREB and produces similar results (synthesis of more PEPCK-C).

Together, cortisol and glucagon can have huge synergistic results, activating the PEPCK-C gene to levels that neither cortisol or glucagon could reach on their own. PEPCK-C is most abundant in the liver, kidney, and adipose tissue.[3]

A collaborative study between the U.S. Environmental Protection Agency (EPA) and the University of New Hampshire investigated the effect of DE-71, a commercial PBDE mixture, on PEPCK enzyme kinetics and determined that in vivo treatment of the environmental pollutant compromises liver glucose and lipid metabolism possibly by activation of the pregnane xenobiotic receptor (PXR), and may influence whole-body insulin sensitivity.[16]

Researchers at Case Western Reserve University have discovered that overexpression of cytosolic PEPCK in skeletal muscle of mice causes them to be more active, more aggressive, and have longer lives than normal mice; see metabolic supermice.

Plants

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PEPCK (EC 4.1.1.49) is one of three decarboxylation enzymes used in the inorganic carbon concentrating mechanisms of C4 and CAM plants. The others are NADP-malic enzyme and NAD-malic enzyme.[17][18] In C4 carbon fixation, carbon dioxide is first fixed by combination with phosphoenolpyruvate to form oxaloacetate in the mesophyll. In PEPCK-type C4 plants the oxaloacetate is then converted to aspartate, which travels to the bundle sheath. In the bundle sheath cells, aspartate is converted back to oxaloacetate. PEPCK decarboxylates the bundle sheath oxaloacetate, releasing carbon dioxide, which is then fixed by the enzyme Rubisco. For each molecule of carbon dioxide produced by PEPCK, a molecule of ATP is consumed.

PEPCK acts in plants that undergo C4 carbon fixation, where its action has been localized to the cytosol, in contrast to mammals, where it has been found that PEPCK works in mitochondria.[19]

Although it is found in many different parts of plants, it has been seen only in specific cell types, including the areas of the phloem.[20]

It has also been discovered that, in cucumber (Cucumis sativus L.), PEPCK levels are increased by multiple effects that are known to decrease the cellular pH of plants, although these effects are specific to the part of the plant.[20]

PEPCK levels rose in roots and stems when the plants were watered with ammonium chloride at a low pH (but not at high pH), or with butyric acid. However, PEPCK levels did not increase in leaves under these conditions.

In leaves, 5% CO2 content in the atmosphere leads to higher PEPCK abundance.[20]

Bacteria

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In an effort to explore the role of PEPCK, researchers caused the overexpression of PEPCK in E. coli bacteria via recombinant DNA.[21]

PEPCK of Mycobacterium tuberculosis has been shown to trigger the immune system in mice by increasing cytokine activity.[22]

As a result, it has been found that PEPCK may be an appropriate ingredient in the development of an effective subunit vaccination for tuberculosis.[22]

Clinical significance

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Activity in cancer

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PEPCK has not been considered in cancer research until recently. It has been shown that in human tumor samples and human cancer cell lines (breast, colon and lung cancer cells) PEPCK-M, and not PEPCK-C, was expressed at enough levels to play a relevant metabolic role.[1][23] Therefore, PEPCK-M could have a role in cancer cells, especially under nutrient limitation or other stress conditions.

Regulation

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In humans

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PEPCK-C is enhanced, both in terms of its production and activation, by many factors. Transcription of the PEPCK-C gene is stimulated by glucagon, glucocorticoids, retinoic acid, and adenosine 3',5'-monophosphate (cAMP), while it is inhibited by insulin.[24] Of these factors, insulin, a hormone that is deficient in the case of type 1 diabetes mellitus, is considered dominant, as it inhibits the transcription of many of the stimulatory elements.[24] PEPCK activity is also inhibited by hydrazine sulfate, and the inhibition therefore decreases the rate of gluconeogenesis.[25]

In prolonged acidosis, PEPCK-C is upregulated in renal proximal tubule brush border cells, in order to secrete more NH3 and thus to produce more HCO3−.[26]

The GTP-specific activity of PEPCK is highest when Mn2+ and Mg2+ are available.[21] In addition, hyper-reactive cysteine (C307) is involved in the binding of Mn2+ to the active site.[10]

Plants

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As discussed previously, PEPCK abundance increased when plants were watered with low-pH ammonium chloride, though high pH did not have this effect.[20]

Classification

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It is classified under EC number 4.1.1. There are three main types, distinguished by the source of the energy to drive the reaction:

References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Phosphoenolpyruvate carboxykinase

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from Grokipedia
Phosphoenolpyruvate carboxykinase (PEPCK) is a critical in the lyase family (EC 4.1.1.32) that catalyzes the reversible and of oxaloacetate (OAA) to form phosphoenolpyruvate (PEP), (GDP), and (CO₂), utilizing (GTP) as the phosphate donor. This reaction represents a rate-limiting step in , enabling the synthesis of glucose from non-carbohydrate precursors such as lactate, , and , primarily in the and to maintain blood glucose levels during or . Mammals express two isoforms of PEPCK: the cytosolic form (PEPCK-C or PCK1), which predominates in gluconeogenic tissues like the liver (accounting for ~90-95% of total activity in rodents and ~50% in humans), and the mitochondrial form (PEPCK-M or PCK2), which is ubiquitously expressed and facilitates direct shuttling of PEP across the mitochondrial membrane to support cytosolic metabolism. Both isoforms share a conserved gene structure with 10 exons and exhibit similar three-dimensional folds, including a nucleotide-binding P-loop and a mobile Ω-loop that regulates substrate access in the active site, as revealed by crystal structures of bacterial and human PEPCK-C. Beyond gluconeogenesis, PEPCK plays essential roles in glyceroneogenesis for triglyceride synthesis in adipose tissue, anaplerosis and cataplerosis in the tricarboxylic acid (TCA) cycle, serine biosynthesis, and even non-metabolic functions such as protein kinase activity in cancer cells. The expression and activity of PEPCK are tightly regulated by hormonal signals, including and glucocorticoids that induce transcription via cAMP-responsive elements, while insulin represses it through FoxO1 and nuclear exclusion; dysregulation of PEPCK is implicated in metabolic disorders like , , and . Evolutionarily conserved across eukaryotes, PEPCK's dual localization allows metabolic flexibility, with the mitochondrial isoform particularly important in species like birds for sustained .

Classification and isoforms

Nomenclature

Phosphoenolpyruvate carboxykinase (PEPCK) is the accepted name for the enzyme that catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate with concomitant phosphorylation, utilizing either GTP or ATP as the phosphate donor. It is also referred to as phosphoenolpyruvate carboxylase or PEP carboxylase, though these terms can cause confusion with the unrelated plant enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which carboxylates phosphoenolpyruvate to oxaloacetate using bicarbonate in C4 photosynthesis. The enzyme's nomenclature reflects its dual action as a carboxy-lyase, classified under the lyase family for its role in cleaving carbon-carbon bonds. The International Union of Biochemistry and (IUBMB) assigns EC 4.1.1.32 to the GTP-dependent form, which predominates in animals and catalyzes the reaction: GTP + oxaloacetate ⇌ GDP + phosphoenolpyruvate + CO₂. The ATP-dependent variant is designated EC 4.1.1.49, with the reaction: ATP + oxaloacetate ⇌ ADP + phosphoenolpyruvate + CO₂, and is more common in , , and some . A less common diphosphate-dependent form exists as EC 4.1.1.38. These classifications emphasize the 's reversible nature, though it primarily functions in the direction of phosphoenolpyruvate formation in . PEPCK was first identified in the 1950s through studies on pigeon liver extracts, where Merton F. Utter and Kiyoshi Kurahashi demonstrated its role in converting oxaloacetate to phosphoenolpyruvate, resolving a key step in . Their seminal work, published in 1953, described the enzyme's activity and partial purification, marking the beginning of understanding its biochemical mechanism. In humans, the cytosolic isoform is encoded by the gene PCK1 (phosphoenolpyruvate carboxykinase 1), located on chromosome 20q13.31, while the mitochondrial isoform is encoded by PCK2 (phosphoenolpyruvate carboxykinase 2) on chromosome 14q11.2. These gene symbols are standardized by the Organisation (HUGO) and reflect the enzyme's compartmentalization, with PCK1 producing the predominant form in liver and for .

Cytosolic isoform (PEPCK-C)

The cytosolic isoform of phosphoenolpyruvate carboxykinase, designated PEPCK-C, is encoded by the PCK1 , which is located on the long arm of human chromosome 20 at position 20q13.31. This gene consists of 10 exons and encodes a protein of 622 with a calculated of approximately 69 kDa. The protein sequence lacks an N-terminal mitochondrial targeting , a feature that distinguishes it from the mitochondrial isoform and directs its exclusive localization to the . In the , PEPCK-C plays a central role in by catalyzing the GTP-dependent of oxaloacetate to form phosphoenolpyruvate, enabling the continuation of glucose synthesis from non-carbohydrate precursors. Expression of PCK1 is prominent in gluconeogenic tissues such as the liver and kidney cortex, as well as in , where it supports . Transcription of the gene is strongly induced during states through hormonal signals like and glucocorticoids, which activate cAMP-responsive elements to elevate PEPCK-C levels and enhance glucose production. The cytosolic isoform exhibits evolutionary conservation across vertebrates, reflecting its essential role in metabolic adaptation to nutrient scarcity. In contrast, some , such as nematodes, predominantly express a mitochondrial form of PEPCK, highlighting clade-specific compartmentalization of the . Sequence analysis reveals that human PEPCK-C shares approximately 70% identity with the mitochondrial isoform (PEPCK-M), underscoring their common catalytic core despite distinct subcellular targeting.

Mitochondrial isoform (PEPCK-M)

The mitochondrial isoform of phosphoenolpyruvate carboxykinase, known as PEPCK-M and encoded by the PCK2 , is located on human chromosome 14q11.2, spanning approximately 16.5 kb from positions 24,094,053 to 24,110,598 on the GRCh38 assembly. The PCK2 produces a precursor protein of 640 with a molecular weight of approximately 71 , featuring an N-terminal mitochondrial targeting sequence (MTS) of about 18 residues that directs the protein to the . Upon import, the MTS is cleaved by mitochondrial processing peptidase, yielding a mature protein of roughly 622 and ~68 that resides in the . PEPCK-M is imported into mitochondria via the canonical presequence pathway, involving recognition by cytosolic chaperones, translocation across the outer membrane through the TOM complex, and passage across the inner membrane via the TIM23 complex, powered by the . This process ensures precise localization to , where the catalyzes the and of oxaloacetate to phosphoenolpyruvate using GTP as the phosphate donor, a reaction essential for integrating mitochondrial metabolism with cytosolic pathways. Unlike the cytosolic isoform, PEPCK-M shares approximately 70% amino acid sequence identity with PEPCK-C but possesses unique adaptations for mitochondrial function, including the MTS. Expression of PCK2 is largely constitutive across most human tissues, with relatively higher levels observed in , , liver, and , reflecting its role in basal metabolic maintenance rather than acute hormonal regulation. In contrast to the highly inducible cytosolic isoform, PEPCK-M shows minimal responsiveness to dietary or hormonal cues, ensuring steady-state activity in energy-demanding tissues like and . A distinctive feature of PEPCK-M is its capacity to generate phosphoenolpyruvate directly within the , facilitating local utilization in processes such as anaplerotic replenishment of TCA cycle intermediates or export to the for and , thereby linking mitochondrial substrate oxidation to broader cellular without relying on malate-aspartate shuttling. This intramitochondrial PEP production supports efficient handling of mitochondrial-derived substrates, such as those from , and contributes to metabolic flexibility in non-gluconeogenic tissues.

Structure

Overall architecture

Phosphoenolpyruvate carboxykinase (PEPCK) is a monomeric composed of a single polypeptide chain that folds into two major structural domains separated by a deep cleft containing the . The N-terminal domain, spanning approximately residues 1–259, adopts an α/β fold responsible for binding phosphoenolpyruvate (PEP) and exhibits structural features akin to nucleotide-binding motifs. The C-terminal domain, encompassing residues 260–622, contains the nucleotide-binding with a fold resembling that of , including a P-loop motif for GTP coordination, and a PEPCK-specific for additional substrate interactions. In the cytosolic isoform (PEPCK-C), structures, determined at resolutions around 2.1–2.3 Å (e.g., PDB entry 1KHG for the apo form and 1KHF for the complex with PEP), reveal a monomeric organization and highlight the conservation of the bilobal architecture across mammalian species. These structures also show coordination of two divalent metal ions at the : Mn²⁺ at the M1 site, which binds the substrate carboxylate groups, and Mg²⁺ at the M2 site, associated with the triphosphate. Both isoforms share this conserved bilobal fold. A key structural feature is the flexible Ω-loop (residues 464–474 in human PEPCK-C), which serves as a lid over the cleft. In its open conformation, the loop is disordered, allowing substrate access; upon binding, it closes to enclose the reaction intermediates, as visualized in crystal structures of substrate-bound forms. This dynamic element is conserved in the shared fold of PEPCK isoforms and is critical for maintaining the integrity of the catalytic pocket.

Species variations

Phosphoenolpyruvate carboxykinase (PEPCK) exhibits significant structural variations across species, reflecting evolutionary adaptations to diverse metabolic needs. Mammalian PEPCK isoforms are GTP-specific and typically comprise around 622 amino acids in humans, featuring a dedicated GTP-binding domain with kinase motifs for nucleotide coordination. In contrast, bacterial PEPCK, such as that from Escherichia coli, is ATP-dependent, 540 amino acids long, and lacks the GTP-binding domain, relying instead on ATP-specific motifs for catalysis. This divergence in nucleotide specificity—GTP in mammals versus ATP in many bacteria—underpins a low sequence identity of less than 20% between these forms, highlighting ancient evolutionary branching despite conserved overall folds like beta-alpha-beta motifs in the nucleotide-binding regions. In , PEPCK is predominantly cytosolic and ATP-dependent, with examples like the enzyme from the alga Scenedesmus obliquus, similar in size to bacterial counterparts but adapted for gluconeogenic roles in lipid mobilization during seed germination. Unlike mammalian forms, PEPCK lacks mitochondrial targeting signals in most cases and instead features regulatory phosphorylation sites that enhance flexibility in carbon flux. Parasitic protozoa, such as , possess a glycosomal isoform of PEPCK, localized to specialized peroxisome-like organelles for compartmentalized , with a compact of 472 and ATP specificity tailored to the parasite's anaerobic . A 2024 CRISPR/Cas9 study confirmed this glycosomal targeting through deletion, revealing unique N-terminal signals that direct the enzyme to these compartments, distinct from cytosolic localization in free-living organisms. Adaptations in extremophiles further diversify PEPCK structure; for instance, a 2025 structural analysis of psychrophilic bacterial PEPCK identified enhanced flexibility in the active-site Ω-loop, facilitating conformational dynamics at low temperatures and enabling efficient in cold environments.

Mechanism

Catalytic reaction

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the committed step of by converting oxaloacetate (OAA) to phosphoenolpyruvate (PEP) through and . The forward reaction, which is essential for glucose synthesis from non-carbohydrate , is given by the equation: \ceoxaloacetate+GTP4−+H+−>phosphoenolpyruvate3−+GDP3−+CO2+H2O\ce{oxaloacetate + GTP^{4-} + H+ -> phosphoenolpyruvate^{3-} + GDP^{3-} + CO2 + H2O} This reaction has a standard free energy change (ΔG°') of approximately +0.8 kJ/mol under physiological conditions (pH 7, 25°C), rendering it nearly reversible and allowing flux in both directions depending on cellular needs. The catalytic mechanism involves two key steps: the β-decarboxylation of OAA to generate a transient enol form of pyruvate, followed by phosphoryl transfer from GTP to the enol oxygen. OAA initially binds to the active site and coordinates directly with a Mn²⁺ ion at the primary metal binding site (M1), which polarizes the C2-C3 bond and promotes CO₂ release, yielding the enolate intermediate stabilized by residues such as Arg-87 (in human PEPCK-C). GTP then binds adjacent to this site via the conserved P-loop (GXXGXGKT/S motif), coordinating a second Mn²⁺ at site M2 to position the γ-phosphate for nucleophilic attack by the enolate oxygen in an Sₙ2 displacement, forming PEP and GDP. Conformational changes briefly close the active site lid to facilitate these steps. Both mammalian isoforms, cytosolic PEPCK-C and mitochondrial PEPCK-M, specifically utilize GTP (or ITP) as the substrate, with Mn²⁺ or Mg²⁺ as essential cofactors. In contrast, PEPCK enzymes from and some other non-mammalian organisms preferentially use ATP, reflecting evolutionary adaptations in nucleotide specificity while conserving the core catalytic architecture.

Conformational changes

Upon binding of GTP to phosphoenolpyruvate carboxykinase (PEPCK), the enzyme undergoes a major conformational transition from an open to a closed state, characterized by a clam-shell-like of the N-terminal domain by approximately 20° relative to the C-terminal domain. This movement narrows the interdomain cleft, positioning the nucleotide-binding site and in optimal alignment for while excluding bulk solvent. A critical aspect of this transition is the ordering of the Ω-loop lid domain, which in the human cytosolic isoform (PEPCK-C) spans residues 464–474. In the open conformation, this loop is disordered and flexible, but GTP binding induces its closure over the , forming stabilizing interactions such as a between Arg-473 and Glu-86 to secure the intermediate during phosphoryl transfer. This lid closure is essential for shielding the reactive enediol intermediate from and ensuring efficient substrate orientation. The loop closure represents the rate-limiting step in the catalytic mechanism, governing the phosphoryl transfer from GTP to the oxaloacetate-derived enediol intermediate. simulations of PEPCK dynamics reveal that these loop movements occur on nanosecond timescales, consistent with the energetic barriers for disorder-to-order transitions in lid-gated enzymes. Although the wild-type enzyme favors the / direction (oxaloacetate to phosphoenolpyruvate), the reverse reaction (phosphoenolpyruvate + CO₂ + GDP to oxaloacetate + GTP) is inefficient due to unfavorable energetics and conformational constraints. However, certain mutants, such as those disrupting the Ω-loop (e.g., lid deletion variants), exhibit residual activity in the reverse direction, albeit reduced by over 10⁶-fold in k_cat, highlighting the 's role in directionality. The Mn²⁺ ion at the primary metal site (M1) plays a pivotal role in stabilizing the enediol intermediate through coordination to its oxygen atoms, with the closed conformation enhancing this geometry. Disruptive mutations near the , such as those affecting residues involved in proton transfer (e.g., analogous to His-232 in bacterial PEPCK), impair loop dynamics and intermediate stabilization, leading to diminished catalytic efficiency. Active site residues like Lys-290 and Arg-87 (in PEPCK-C) are repositioned by these changes to interact with the substrates and metals.

Functions

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a key irreversible step in , converting oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the through and , utilizing GTP as the energy source. This reaction, primarily mediated by the cytosolic isoform PEPCK-C, occurs in the liver and and effectively bypasses the irreversible step of , enabling the synthesis of glucose from non-carbohydrate precursors. The overall process requires mitochondrial OAA to be shuttled to the , often as malate via the malate-aspartate shuttle, before being reconverted to OAA for PEPCK action. Substrates for this pathway include lactate, which is oxidized to pyruvate and then carboxylated to OAA by in the mitochondria, as well as , which is transaminated to pyruvate, and various tricarboxylic acid (TCA) cycle intermediates that generate OAA. These precursors are essential during , when stores are depleted, allowing the liver to maintain blood glucose levels through . PEPCK's position ensures that the energy-intensive conversion from OAA to PEP drives the forward flux toward glucose production. As a rate-limiting enzyme, PEPCK exerts significant control over gluconeogenic flux; studies using liver-specific PEPCK-C knockout mice demonstrate that hepatic glucose output from lactate and pyruvate is completely abolished, underscoring its indispensable role in this pathway. Although total hepatic glucose production during fasting can be partially sustained via alternative routes like glycerol, the enzyme's activity is critical for the majority of gluconeogenesis. In the fasting liver, substrates requiring PEPCK—such as lactate and alanine—account for approximately 90% of gluconeogenic flux, highlighting its quantitative dominance in glucose homeostasis. Its expression is rapidly induced by glucagon during fasting to enhance this capacity.

Glyceroneogenesis and serine biosynthesis

PEPCK-C also plays a key role in , the synthesis of glycerol-3-phosphate from non-carbohydrate precursors in , which is essential for storage and mobilization. By converting cytosolic OAA to PEP, PEPCK provides a carbon source for (DHAP) production via and , supporting during feeding and re-esterification of free fatty acids post-lipolysis. Dysregulation of this pathway contributes to and . In addition, PEPCK, particularly the mitochondrial isoform PEPCK-M, supports serine biosynthesis by generating PEP from TCA-derived OAA, which can be converted to 3-phosphoglycerate for entry into the phosphorylated pathway of serine synthesis. This function is crucial in anabolic states or nutrient-limited conditions, such as in cancer cells relying on for , where PEPCK maintains flux through serine and one-carbon metabolism.

Cataplerosis and anaplerosis

The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the PCK2 gene, plays a crucial role in cataplerosis by converting excess oxaloacetate (OAA) derived from the tricarboxylic acid (TCA) cycle into phosphoenolpyruvate (PEP), thereby preventing the accumulation of TCA intermediates that could inhibit cycle flux. This cataplerotic function is essential in tissues with high metabolic demands, such as the liver and kidney, where PEPCK-M helps maintain TCA cycle homeostasis by exporting OAA for alternative metabolic pathways. Recent studies have shown that loss of the cytosolic isoform PEPCK-C (encoded by PCK1) disrupts cataplerosis in kidney tubular cells, leading to mitochondrial dysfunction characterized by reduced respiration and buildup of TCA metabolites like OAA and malate. In the context of anaplerosis reversal, PEPCK-mediated cataplerosis indirectly supports the replenishment of TCA intermediates; the PEP generated can be decarboxylated to pyruvate by , which is then carboxylated by to reform OAA, facilitating cycling and balance of TCA pool levels. This mechanism ensures dynamic flux without net loss of intermediates during periods of high anaplerotic input from or other sources. PEPCK's cataplerotic activity is particularly critical in the , where it contributes to acid-base balance by supporting from TCA-derived substrates and maintaining lactate homeostasis through efficient mitochondrial metabolism. In renal tubular cells, PCK1 deficiency impairs these processes, exacerbating acidosis-induced and disrupting lactate clearance. Dysregulated cataplerosis via PCK1 loss promotes progression, as evidenced by a 2025 study demonstrating that Pck1 deletion in tubular cells causes mitochondrial , inflammation, and through blocked TCA efflux and accumulated intermediates. Restoring PCK1 expression in these models mitigates and preserves renal function, highlighting its protective role against fibrotic remodeling in .

Roles in non-mammalian organisms

In , cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in C4 and (CAM) photosynthesis by facilitating the supply of phosphoenolpyruvate (PEP) through the of oxaloacetate (OAA) derived from C4 acids. In PEPCK-type C4 , such as certain grasses, this operates in bundle sheath cells, where it converts OAA to PEP and CO2, enabling efficient carbon fixation and minimizing by concentrating CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase. In CAM , like succulents, PEPCK contributes to daytime of stored malate (a C4 acid) to regenerate PEP, supporting the release of CO2 for the while conserving water through nocturnal CO2 uptake. This localization and function highlight PEPCK's adaptation to specialized photosynthetic pathways that enhance carbon assimilation under arid or high-light conditions. Overexpression of the maize ZmPCK2 gene, encoding a cytosolic PEPCK isoform, has been shown to improve in transgenic by enhancing metabolic flexibility and stress response. Under water-deficit conditions, ZmPCK2-overexpressing lines exhibited reduced oxidative damage, maintained higher , and showed upregulated genes involved in synthesis, leading to better survival and yield compared to wild-type plants. This demonstrates PEPCK's potential in engineering stress-resilient crops, particularly in C4 species like where it supports PEP regeneration for sustained carbon flow during environmental stress. In bacteria, PEPCK (often denoted as PckA) is essential for , converting TCA cycle intermediates like OAA to PEP to support the synthesis of glucose and other carbohydrates from non-sugar sources. In Escherichia coli, this enzyme enables growth on TCA-derived substrates by replenishing PEP for biosynthetic pathways, with its activity tightly regulated to balance anaplerotic and gluconeogenic fluxes during carbon-limited conditions. In the pathogen Mycobacterium tuberculosis, PEPCK serves as a key by sustaining from host-derived fatty acids, allowing the bacterium to persist within macrophages and evade immune clearance through metabolic dormancy and intracellular survival. In parasites such as , glycosomal PEPCK is vital for energy metabolism during the life cycle, particularly in providing ATP and glycolytic intermediates needed for host . CRISPR/Cas9-mediated knockout of the glycosomal PEPCK gene impairs metacyclogenesis (differentiation into infective forms) in the insect vector and reduces infectivity in vertebrate hosts, as the enzyme links to for sustaining energy demands under nutrient-scarce conditions inside cells. Evolutionarily, ATP-dependent PEPCK variants in anaerobic bacteria represent adaptations for fermentation pathways, differing from the GTP-dependent forms prevalent in aerobes and eukaryotes. In anaerobes like Propionibacterium freudenreichii, this isoform facilitates the conversion of PEP to OAA in succinate-propionate fermentation, conserving ATP and enabling growth on complex substrates without oxygen, thus supporting niche colonization in oxygen-depleted environments.

Regulation

Transcriptional regulation

The promoter region of the PCK1 gene, which encodes the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C), contains multiple regulatory elements that control its transcription in mammals, particularly in gluconeogenic tissues. Key among these are the cAMP response element (CRE), which facilitates induction by glucagon and cyclic AMP (cAMP) through binding of the transcription factor CREB; the glucocorticoid response element (GRE), which mediates activation by glucocorticoids via the glucocorticoid receptor (GR); and the thyroid hormone response element (T3RE), which responds to thyroid hormone (T3) to enhance expression. These elements enable rapid adjustments in PCK1 transcription in response to hormonal signals that promote gluconeogenesis during fasting. Hormonal regulation of PCK1 transcription is tightly coordinated, with insulin acting as a by promoting and nuclear exclusion of the FoxO1, thereby inhibiting its binding to the promoter and reducing PCK1 expression. In contrast, induce transcription through direct GR binding to the GRE within the glucocorticoid response unit (), often in cooperation with accessory factors like CREB to amplify the response. Thyroid hormone further potentiates this by binding to the T3RE, synergizing with cAMP and signals to elevate PCK1 levels in the liver. Tissue-specific expression of PCK1 is governed by enhancers that confer liver- and kidney-enriched activity, including DNase I-hypersensitive sites that form distinct structures in these organs compared to non-gluconeogenic tissues. In the , specific hypersensitive sites (e.g., HSS A and others) interact with renal-enriched factors to drive expression, while liver enhancers involve hepatocyte nuclear factor-1 (HNF-1) binding for basal and inducible activity. Additionally, circadian rhythms modulate PCK1 transcription through clock genes like cryptochromes (CRY1 and CRY2), which rhythmically repress the promoter via interaction with the GR at the GRE, linking metabolic output to daily cycles. Genetic variants in PCK1 influence its transcriptional and are associated with risk. Notably, the promoter SNP -232C>G enhances basal and cAMP-mediated transcription, increasing PCK1 expression and conferring susceptibility to in multiple populations, with odds ratios up to 2.8 in Caucasians.

Allosteric and post-translational control

Phosphoenolpyruvate carboxykinase (PEPCK) is subject to allosteric and post-translational that allows rapid modulation of its activity in response to metabolic demands, independent of changes in . Although mammalian PEPCK isoforms lack classical allosteric effectors like those in some bacterial or enzymes, the is sensitive to substrate and product concentrations, with GTP acting as a substrate that drives the forward reaction toward phosphoenolpyruvate (PEP) formation, effectively activating when GTP levels are high. Conversely, PEP exerts product inhibition in the oxaloacetate (OAA)-to-PEP direction, providing feedback to limit flux when downstream glycolytic intermediates accumulate. The also exhibits sensitivity, with optimal activity in the physiological range of 7.0-7.5, enabling indirect by cellular acidification or alkalization during metabolic stress. Kinetic parameters further contribute to this control, with reported Km values for OAA of approximately 0.04 mM and for GTP of approximately 0.07 mM in mammalian PEPCK, placing the near saturation under typical physiological substrate concentrations and allowing fine-tuning by small fluctuations in metabolite levels. Natural inhibitors such as 3-mercaptopicolinic acid (3-MPA) bind allosterically to a pocket in the monomeric form of PEPCK, inducing a conformational change that disrupts the nucleotide-binding site and potently inhibits catalysis, with potential implications for therapeutic targeting of . This allosteric mechanism highlights how exogenous or endogenous small molecules can rapidly suppress PEPCK activity to coordinate with tricarboxylic acid (TCA) cycle flux. Post-translational modifications provide another layer of immediate control, primarily affecting protein stability and turnover. In the cytosolic isoform (PEPCK-C or PCK1), lysine promotes ubiquitination and proteasomal degradation, reducing abundance and suppressing during nutrient-replete conditions; this process is reversed by deacetylation via SIRT1, which enhances stability and sustains activity. further modulates this balance, as GSK3β-mediated at specific sites impairs SIRT1 deacetylation efficiency, thereby accelerating degradation and linking PEPCK levels to insulin signaling pathways. These modifications ensure that existing PEPCK molecules are rapidly adjusted without requiring new protein synthesis, contrasting with slower transcriptional mechanisms. For the mitochondrial isoform (PEPCK-M or PCK2), post-translational regulation is less well-defined but likely involves similar dynamics within the mitochondrial acetylome, influenced by sirtuins like SIRT3 to maintain metabolic flexibility in anaplerotic and cataplerotic roles. Overall, these controls integrate PEPCK into broader cellular , preventing futile cycling with the TCA cycle and responding to energy status.

Clinical significance

In cancer

Phosphoenolpyruvate carboxykinase (PEPCK), encoded by PCK1 and PCK2, is frequently upregulated in various cancers, including lung and liver malignancies, where it facilitates metabolic adaptation by supporting gluconeogenesis-like pathways to generate biomass precursors under nutrient stress conditions such as hypoxia. In lung cancer cells, mitochondrial PEPCK (PCK2) overexpression enables the conversion of glutamine-derived oxaloacetate to phosphoenolpyruvate, replenishing glycolytic intermediates essential for anabolic processes when glucose is limited. Similarly, in hepatocellular carcinoma, elevated PCK1 levels promote gluconeogenic flux to counteract hypoxic suppression of glycolysis, sustaining tumor proliferation. PCK2 plays a key role in metabolic rewiring, particularly in non-small cell lung cancer (NSCLC), where it confers resistance to during glucose deprivation by maintaining mitochondrial function and inhibiting activation. This adaptation allows NSCLC cells to utilize alternative carbon sources like for energy and , thereby promoting tumorigenesis and survival in nutrient-poor tumor microenvironments. As a therapeutic target, PEPCK inhibition has shown promise in reducing tumor growth; for instance, pharmacological blockade with 3-mercaptopicolinate or genetic silencing of PCK2 enhances in low-glucose conditions and suppresses NSCLC xenograft progression . High PEPCK expression correlates with poor clinical across multiple cancers and is associated with somatic mutations in PCK1 and PCK2 that drive oncogenic signaling. In the tumor immune context, elevated PEPCK levels are linked to reduced T-cell infiltration, potentially fostering an immunosuppressive microenvironment that aids immune escape in lung adenocarcinoma. This association underscores PEPCK's role in modulating antitumor immunity, with higher expression correlating to lower densities of + T cells and poorer immune responsiveness.

In metabolic and kidney diseases

In metabolic disorders such as and , dysregulation of PCK1 (the encoding cytosolic phosphoenolpyruvate carboxykinase, or PEPCK-C) in plays a significant role in . Overexpression of PCK1 in adipocytes enhances , leading to increased re-esterification of fatty acids derived from and promoting fat storage, which contributes to without initial . However, under high-fat diet conditions, this overexpression impairs adipose lipid buffering capacity, exacerbating and systemic metabolic dysfunction. Conversely, adipose-specific PCK1 knockout in mice results in due to reduced and synthesis, rendering them resistant to diet-induced by limiting fat accumulation. In kidney diseases, PCK1 deficiency disrupts mitochondrial cataplerosis in proximal tubular cells, leading to tricarboxylic acid (TCA) cycle blockade, accumulation of metabolites, and mitochondrial injury characterized by swelling, cristae loss, and reduced ATP production. This mitochondrial dysfunction triggers inflammation, tubular injury (marked by elevated KIM-1 expression), and renal fibrosis, accelerating progression to chronic kidney disease (CKD) and increasing mortality in models of ischemia-reperfusion and cisplatin-induced injury. PCK1 expression inversely correlates with mitochondrial defects in human CKD, and its restoration improves glomerular filtration rate, reduces fibrosis scores, and limits proinflammatory responses. In renal mitochondria, PCK1 supports cataplerosis to maintain TCA cycle flux, indirectly aiding ammoniagenesis during metabolic stress. The mitochondrial isoform of PEPCK (PEPCK-M, encoded by PCK2) in proximal tubules is crucial for metabolism and ammoniagenesis, key processes in renal acid-base . During , is deaminated in mitochondria to form α-ketoglutarate, which enters the TCA cycle; PEPCK-M then converts oxaloacetate to phosphoenolpyruvate, facilitating the release of equivalents to buffer systemic while generating for urinary excretion. Impaired PEPCK activity reduces ammoniagenesis, contributing to acid-base imbalances in CKD. Recent studies highlight PCK1's protective role in specific renal conditions, such as . In human exposed to polymeric IgA1, PCK1 expression is downregulated, promoting and fibrotic progression; however, PCK1 overexpression inhibits these effects, reducing proinflammatory release and deposition to mitigate cellular .

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