Phosphoglycerate kinase

Phosphoglycerate kinase (PGK; EC 2.7.2.3) is a glycolytic enzyme that catalyzes the reversible phosphoryl transfer from 1,3-bisphosphoglycerate to ADP, producing 3-phosphoglycerate and ATP.^[1] This reaction represents the first ATP-generating step in glycolysis and plays a crucial role in cellular energy production across Bacteria, Archaea, and Eukarya.^[1] The enzyme is highly conserved evolutionarily, reflecting its fundamental importance in carbohydrate metabolism.^[1] Structurally, PGK is typically a monomeric protein of about 45 kDa, featuring a bilobal architecture with N- and C-terminal domains connected by a flexible hinge region that enables large-scale conformational changes between open and closed states during catalysis.^[1] These dynamics facilitate substrate binding and product release, ensuring efficient phosphoryl transfer.^[1] In eukaryotes, including humans, the primary isoform is encoded by the PGK1 gene located on the X chromosome, with ubiquitous expression in tissues such as the kidney and heart.^[2] Beyond glycolysis, PGK exhibits moonlighting functions, such as acting as a disulfide reductase and contributing to processes like DNA replication and angiogenesis in tumor cells.^[2] Deficiency in PGK, often due to mutations in PGK1, results in a rare X-linked disorder characterized by chronic hemolytic anemia, myopathy, and neurological impairments, underscoring the enzyme's essential role in energy metabolism.^[3] Additionally, PGK's involvement in cancer progression has emerged as a notable aspect, with elevated expression in tumors promoting metabolic adaptation and metastasis.^[4]

Overview and Nomenclature

Enzyme Classification

Phosphoglycerate kinase is classified under EC 2.7.2.3 as a phosphotransferase in the sub-subclass 2.7.2, which specifically catalyzes the reversible transfer of a high-energy phosphate group from the acyl phosphate of 1,3-bisphosphoglycerate (1,3-BPG) to ADP.^[5] The catalyzed reaction follows the equation:

\text{1,3-bisphosphoglycerate} + \text{[ADP](/page/ADP)} \rightleftharpoons \text{3-phosphoglycerate} + \text{ATP}

This phosphotransfer is reversible, with the forward direction predominating in glycolysis to generate ATP via substrate-level phosphorylation and the reverse direction occurring in gluconeogenesis to consume ATP for the synthesis of 1,3-BPG; the stoichiometry is equimolar (1:1) for all substrates and products, requiring Mg^{2+} as a cofactor to stabilize the nucleotide triphosphate.^[5]^[6] The enzyme was first isolated and crystallized from yeast in 1947, playing a key role in the mid-20th century elucidation of the Embden-Meyerhof glycolytic pathway.76966-X/pdf) In human PGK1, representative kinetic parameters demonstrate high substrate affinity, with Km values of approximately 0.007 mM for 1,3-BPG and 0.008 mM for ADP under physiological conditions.48506-1/fulltext)

Gene and Protein Basics

Phosphoglycerate kinase in humans is encoded by two distinct genes: PGK1 and PGK2. The PGK1 gene, located on the long arm of the X chromosome at Xq21.1, is ubiquitously expressed across tissues and produces a transcript with a 1,251 bp coding sequence that encodes a 417-amino acid protein with a molecular mass of approximately 44.6 kDa.^[2]^[6] This isoform supports essential glycolytic functions in somatic cells and various other cellular processes. In contrast, the PGK2 gene resides on the short arm of chromosome 6 at 6p12.3 and is expressed specifically in testicular germ cells during spermatogenesis, featuring a similar 1,251 bp coding sequence that yields a 417-amino acid protein.^[7]^[8] The PGK2 protein exhibits 88% amino acid sequence identity to PGK1, reflecting their shared evolutionary origin through retrotransposition, yet it is adapted for specialized roles in male reproductive tissues.^[9] The amino acid sequences of phosphoglycerate kinase proteins demonstrate high conservation across eukaryotic species, underscoring their fundamental role in energy metabolism. For instance, human PGK1 shares approximately 70% sequence identity with the orthologous enzyme from the yeast Saccharomyces cerevisiae, highlighting preserved structural and functional elements despite evolutionary divergence.^[10] This conservation extends to key catalytic residues and overall domain architecture, enabling comparable enzymatic activity in diverse organisms. Physicochemically, phosphoglycerate kinase functions as a monomeric enzyme, lacking the need for multimeric assembly to achieve catalytic competence.^[11] The human PGK1 isoform has an isoelectric point of approximately 8.3, which contributes to its solubility in physiological buffers and stability under neutral pH conditions (pH 6.5–8.0).^[12]^[13] These properties facilitate its high intracellular abundance and resistance to denaturation in cytosolic environments, with the protein remaining soluble and active in dilute aqueous solutions without aggregation. Significant milestones in the genetic characterization of phosphoglycerate kinase occurred post-2000 through advancements in genomic sequencing. The PGK1 gene was fully integrated into the reference human genome assembly as part of the Human Genome Project, which achieved a complete draft sequence in 2003, enabling precise mapping and annotation of its regulatory elements and variants.^[14] This integration has supported subsequent studies on disease-associated mutations and expression patterns, building on earlier cDNA sequencing efforts.

Biological Role

Phosphoglycerate kinase (PGK) occupies a central position in glycolysis as the enzyme catalyzing step 7, where it facilitates substrate-level phosphorylation by transferring the acyl phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, yielding 3-phosphoglycerate (3-PG) and ATP.^[1] This reaction is highly conserved across organisms and represents one of the two ATP-generating steps in the glycolytic pathway, with the enzyme operating near equilibrium to maintain flux efficiency.^[15] In the context of complete glucose oxidation, the two PGK reactions per glucose molecule (due to the bifurcation of glyceraldehyde-3-phosphate) contribute 2 ATP molecules to the net yield of glycolysis.^[16] Upstream, glyceraldehyde-3-phosphate dehydrogenase generates the high-energy 1,3-BPG intermediate using NAD+ and inorganic phosphate, while downstream, phosphoglycerate mutase converts 3-PG to 2-PG, linking PGK directly to subsequent dehydration and enolase steps in the payoff phase of glycolysis.^[1] The reaction catalyzed by PGK is reversible, enabling its role in gluconeogenesis, where it consumes ATP to phosphorylate 3-PG into 1,3-BPG, thereby supporting the synthesis of glucose from non-carbohydrate precursors such as lactate or amino acids.^[1] This reverse flux is particularly prominent in conditions of glucose scarcity, where PGK helps reverse the glycolytic direction to replenish glycogen stores or maintain blood glucose levels, with the enzyme's kinetic properties favoring the gluconeogenic direction under high ATP/ADP ratios.^[1] In mammalian liver and kidney tissues, this reversibility ensures metabolic flexibility, integrating PGK into the broader gluconeogenic pathway alongside enzymes like fructose-1,6-bisphosphatase.^[1] In photosynthetic organisms, PGK plays an analogous but reversed role in the Calvin-Benson-Bassham cycle within chloroplasts, where it phosphorylates 3-PG to 1,3-BPG using ATP, facilitating the reduction phase of carbon fixation following Rubisco-mediated CO2 incorporation.^[17] This plastidial isoform, often redox-regulated by thioredoxin to align with light-dependent ATP availability, supports the assimilation of atmospheric CO2 into triose phosphates.^[17] In leaves, the flux through chloroplastic PGK in the Calvin cycle dominates, accounting for 90-95% of total PGK activity and exceeding glycolytic flux by approximately 10-20 times, reflecting the high demand for carbon fixation during photosynthesis compared to respiratory breakdown in the cytosol.^[17] This integration underscores PGK's versatility in balancing anabolic and catabolic carbon flows across related pathways.^[1]

Non-Glycolytic Functions

Beyond its canonical role in glycolysis, phosphoglycerate kinase (PGK), particularly the PGK1 isoform, exhibits diverse moonlighting functions that extend to nucleic acid metabolism, signaling, and anti-angiogenic processes. These non-glycolytic activities highlight PGK's versatility as a multifunctional protein, conserved across evolutionary domains from bacteria to humans, where structural adaptations enable additional roles without altering the core enzyme sequence.^[1] One prominent non-glycolytic function of extracellular PGK1 is its thiol reductase activity, where it reduces disulfide bonds in plasmin to generate angiostatin, a potent inhibitor of angiogenesis that suppresses tumor vascularization and growth. This process involves PGK1's cysteine residues facilitating the cleavage of specific plasmin kringle domains, leading to anti-tumor effects observed in models of prostate and other cancers. Seminal studies demonstrated that tumor-secreted PGK1 elevates plasma angiostatin levels in vivo, reducing tumor burden in mice, underscoring its therapeutic potential in inhibiting pathological neovascularization.^[18]^[19] In nucleic acid metabolism, nuclear-localized PGK1 contributes to DNA replication and repair by acting as a cofactor in primer recognition complexes. Specifically, PGK1 forms part of the primer recognition protein (PRP) with annexin II, binding to DNA polymerase α to facilitate lagging-strand synthesis during replication. Additionally, PGK1 interacts with the CDC7 kinase at replication origins, converting ADP to ATP to relieve ADP-mediated inhibition of CDC7, thereby promoting origin firing and DNA synthesis; this mechanism is implicated in tumorigenesis, as nuclear PGK1 overexpression enhances replication in brain cancer models. These roles position PGK1 as a metabolic sensor in the nucleus, linking glycolytic intermediates to genome stability.^[1]30754-8)^[20] PGK1 also participates in signal transduction and apoptosis regulation in cancer cells, where it phosphorylates substrates like PRAS40 to activate AKT/mTOR pathways, inhibiting autophagy and promoting cell survival. Overexpression of PGK1 reduces apoptosis in various malignancies, while its depletion sensitizes cells to death signals, reversible by autophagy blockers, thus linking metabolic flux to anti-apoptotic signaling. Recent findings (2023–2025) further reveal PGK1's role in viral infections, where it stimulates β-catenin-dependent transcription to enhance replication; for instance, in bovine herpesvirus 1 (BoHV-1), PGK1 upregulation during infection boosts β-catenin activity, increasing viral gene expression and progeny yield, a mechanism potentially conserved in other pathogens.^[19]^[21] The moonlighting functions of PGK are evolutionarily conserved, with the enzyme's core structure maintained from bacterial ancestors to human isoforms, allowing neofunctionalization such as disulfide reduction and DNA binding without loss of catalytic efficiency. In kinetoplastids like Trypanosoma brucei, duplicated PGK genes encode variants with additional domains (e.g., helix-turn-helix for potential DNA regulation), reflecting adaptations to parasitic lifestyles, while in eukaryotes, this conservation enables coordinated metabolic and non-metabolic responses across cellular compartments.^[1]^[11]

Molecular Structure

Domain Organization

Phosphoglycerate kinase (PGK) is characterized by a bilobal monomeric structure, with the N-terminal domain spanning residues 1–220 and adopting a canonical Rossmann fold topology consisting of a central β-sheet flanked by α-helices; this domain accommodates the binding of 3-phosphoglycerate (3-PG). The C-terminal domain, encompassing residues 250–417, features an α/β fold with a nucleotide-binding site for substrates such as ATP or ADP, also incorporating elements of a Rossmann-like motif. These domains are connected by a flexible hinge region comprising residues ~221–249, which permits large-scale hinge-bending motions to facilitate substrate alignment during catalysis.^[1]^[22]^[23] The overall architecture positions the substrate-binding sites at opposite ends of an inter-domain cleft in the open conformation, with the hinge enabling closure to approximate the distant active sites. Crystal structures confirm this organization, with the inaugural determination of horse muscle PGK in 1972 at 6 Å resolution revealing the two distinct globular lobes separated by the cleft. Subsequent high-resolution structures of human PGK1, such as the 2.15 Å resolution model (PDB: 3C39), delineate the precise domain interfaces and underscore the conservation of this layout across species.^[24]^[25]^[26] Stabilization of the monomer relies on a hydrophobic core at the domain interface, involving non-polar residues that maintain structural integrity without promoting oligomerization; PGK exists predominantly as a monomer in solution, as evidenced by biochemical and crystallographic analyses. This modular design supports the enzyme's function while allowing dynamic flexibility.^[1]^[27]

Key Structural Features

Phosphoglycerate kinase (PGK) harbors Mg²⁺ ions in its active site, essential for facilitating the phosphoryl transfer reaction. The Mg²⁺ ion coordinates the β-phosphate of ADP, primarily through interactions with aspartate and threonine residues, such as Asp373 and Thr378 in the human enzyme, which help position the nucleotide substrate within the C-terminal domain. A second Mg²⁺ ion stabilizes the transferring phosphate group of 1,3-bisphosphoglycerate (1,3-BPG), coordinated by conserved residues including Glu332 and water molecules, ensuring proper alignment for catalysis and neutralizing negative charges during the transition state. These divalent cations are conserved across PGK orthologs and are critical for the enzyme's efficiency, as their absence significantly impairs activity.^[28]^[1] Central to the active site's functionality are several conserved residues that orchestrate substrate binding and catalysis. Lys219, located in the catalytic loop, serves as an electrophilic catalyst by neutralizing the developing negative charge on the transferring phosphate through hydrogen bonding in the closed conformation. Complementing this, Arg38 and Arg65 in the N-terminal domain position the carboxylate and phosphate groups of 3-phosphoglycerate (3-PG) or 1,3-BPG via electrostatic interactions, stabilizing the substrates prior to domain closure. These residues form a basic patch that excludes water and promotes the hinge-bending motion, bringing the distant substrate-binding sites into proximity (~4 Å) for direct phosphoryl transfer without a covalent enzyme-substrate intermediate. Mutations in these residues, such as R65Q, disrupt substrate positioning and reduce catalytic efficiency.^[29]^[30] Glycosylation patterns differ markedly between prokaryotic and eukaryotic PGK variants, reflecting evolutionary adaptations. Prokaryotic PGK lacks glycosylation sites, maintaining a simple cytoplasmic role, whereas eukaryotic PGK1 exhibits post-translational modifications, including O-GlcNAcylation at sites like Thr255, which modulates enzymatic activity, mitochondrial translocation, and coordination between glycolysis and the TCA cycle. While N-glycosylation is not prominent in the core human PGK1 sequence, certain isoforms or paralogs in eukaryotes may feature potential N-linked sites, such as near Asn27, influencing protein stability or interactions in glycosomal compartments of organisms like trypanosomes. These modifications are absent in bacterial counterparts, highlighting domain-specific regulatory mechanisms.^[31]^[1]

Catalytic Mechanism

Reaction Steps

The catalytic reaction of phosphoglycerate kinase (PGK) proceeds through a series of ordered steps involving substrate binding, conformational rearrangement, phosphoryl transfer, and product release. In the forward direction of glycolysis (1,3-bisphosphoglycerate [1,3-BPG] + ADP → 3-phosphoglycerate [3-PG] + ATP), the enzyme in its open conformation first binds 1,3-BPG at the N-terminal domain, followed by Mg²⁺-complexed ADP at the C-terminal domain; in the reverse direction, 3-PG binds first, followed by Mg²⁺-ATP.^[32] This sequential binding induces a large hinge-bending motion between the two domains, closing the active site cleft by approximately 26° and excluding solvent to prevent unproductive hydrolysis.^[33] The closure aligns the substrates optimally, with the C-terminal domain's nucleotide-binding site and the N-terminal domain's acyl phosphate site now in close proximity (inter-domain distance reduced from ~20 Å to ~5 Å).^[34] In the closed conformation, the β-phosphate oxygen of ADP launches a nucleophilic attack on the C1 carbonyl carbon of 1,3-BPG, transferring the phosphoryl group from the C1 acyl phosphate of 1,3-BPG to ADP, with release of the 3-phosphoglycerate carboxylate. This step is facilitated by Lys219, which stabilizes the pentacoordinate transition state through hydrogen bonding and electrostatic interactions, neutralizing charge buildup; Mg²⁺ ions further stabilize the negatively charged phosphates.^[34] The phosphoryl transfer occurs via an associative SN2-like mechanism, characterized by inversion of configuration at the phosphorus atom, ensuring stereospecific transfer to the pro-R oxygen of ADP. Isotope exchange experiments using ¹⁸O-labeled substrates confirm direct in-line transfer of the phosphate without dissociation to a free metaphosphate intermediate or formation of a covalent phosphoenzyme adduct. Upon completion of phosphoryl transfer, the products ATP and 3-PG occupy the closed active site briefly before the enzyme reopens via reversal of the hinge-bending motion. Product release follows the transition to the open conformation, with ATP dissociating from the C-terminal domain and 3-PG from the N-terminal domain. This domain-opening step is rate-limiting for the overall cycle, as the enzyme spends over 90% of its time in the open state, limiting the turnover number to k_cat ≈ 950 s⁻¹ for human PGK1 under physiological conditions.^[33]^[35]

Transition State and Energetics

The transition state in the phosphoglycerate kinase (PGK)-catalyzed reaction features a metastable intermediate with significant charge buildup on the transferring phosphate group from 1,3-bisphosphoglycerate to ADP. This negative charge is primarily stabilized by positively charged arginine residues, such as Arg38 and Arg65 in the human enzyme, which form salt bridges with the phosphate, alongside coordination by Mg²⁺ ions that bridge the β-γ phosphates of ATP/ADP and the substrate carboxyl group. These interactions lower the activation free energy barrier (ΔG‡) to approximately 15 kcal/mol, enabling efficient phosphoryl transfer at physiological rates.^[36]^[30] The overall free energy profile of the PGK reaction is near-equilibrium under cellular conditions (ΔG ≈ 0 kJ/mol), reflecting its reversible nature in glycolysis, though the standard free energy change (ΔG°') favors the forward direction at -16.2 ± 0.2 kJ/mol (pH 7, 37°C). This thermodynamic favorability is coupled to the exergonic ATP hydrolysis in upstream and downstream steps, maintaining glycolytic flux despite the reaction's intrinsic reversibility.^[37] Quantum mechanics/molecular mechanics (QM/MM) simulations of the PGK active site highlight the critical role of Lys219, whose protonated side chain positions near the transferring phosphate to delocalize developing negative charge via electrostatic stabilization, thereby reducing the activation barrier by an estimated 10-15 kcal/mol relative to the solution-phase reaction. These computations, often employing density functional theory for the quantum region, confirm an associative SN2-like transition state.^[30] PGK activity is pH-dependent, with optimal catalysis occurring between pH 7 and 8, where protonation states of the substrates (e.g., deprotonated 1,3-bisphosphoglycerate carboxylate) and active-site residues like His167 facilitate nucleophilic attack and charge stabilization without excessive protonation hindering Mg²⁺ binding.^[38] Studies on PGK1 mutants reveal perturbed energetics with ΔΔG values of 5-10 kcal/mol, reflecting reduced thermodynamic and kinetic stability that impairs catalytic efficiency. For instance, mutations like p.Val216Glu destabilize the folded state, increasing unfolding propensity and altering the energy landscape for domain closure essential to catalysis.^[39]^[40]

Regulation

Environmental Modulators

PGK exhibits optimal activity under physiological conditions mimicking mammalian cellular environments, with peak performance at 37°C and pH 7.0. The enzyme maintains thermal stability up to approximately 50°C, beyond which reversible inactivation occurs due to partial unfolding of its two-domain structure, although full denaturation requires higher temperatures.^[41] These optima ensure efficient function in glycolysis within human cells, where deviations in pH or temperature—such as during fever or acidosis—can reduce activity by 20-40%. In vivo, macromolecular crowding by cellular macromolecules like proteins and metabolites significantly enhances PGK activity compared to dilute in vitro conditions. Crowding agents such as Ficoll at 100-200 mg/mL increase enzymatic turnover approximately 5-fold at 100 mg/mL and over 12-fold (viscosity-adjusted) at 200 mg/mL by compacting the enzyme's open conformation, reducing the interdomain distance and facilitating substrate access without requiring full hinge-bending motion.^[42] This effect mimics the dense cytosolic environment (occupying ~30% volume), promoting a more catalytically competent state. Certain small-molecule inhibitors, such as salicylates, further exemplify environmental modulation by acting as substrate mimics. Salicylates reduce the maximum velocity (V_max) of yeast PGK through competitive binding at the nucleotide site in the C-domain, with inhibition constants around 10 mM for salicylate itself.^[43] This mimicry disrupts ATP coordination, providing a model for how therapeutic compounds might target PGK in metabolic disorders.

Cellular Control Mechanisms

Phosphoglycerate kinase 1 (PGK1) undergoes several post-translational modifications that fine-tune its enzymatic activity, localization, and stability within cells. Acetylation at lysine 323 (K323) significantly enhances PGK1's catalytic efficiency, thereby boosting glycolytic flux, glucose uptake, ATP production, and lactate output, which collectively support cell proliferation and metastasis in liver cancer.^[44] Phosphorylation at serine 203 (Ser203) by extracellular signal-regulated kinase 1/2 (ERK1/2), triggered by hypoxia, epidermal growth factor receptor (EGFR) activation, or oncogenic mutations like K-Ras/B-Raf, shifts PGK1 toward favoring the Warburg effect and promotes tumorigenesis in glioblastoma by altering its metabolic output.^[45] Similarly, phosphorylation at threonine 243 (Thr243) by 3-phosphoinositide-dependent protein kinase 1 (PDPK1), often induced by interleukin-6 from M2 macrophages, increases PGK1's affinity for substrates like 3-phosphoglycerate, thereby accelerating aerobic glycolysis and enhancing tumor cell migration and invasion.^[46] Protein-protein interactions further modulate PGK1's function in cellular metabolism and stress responses. PGK1 associates with the molecular chaperone heat shock protein 90 (HSP90), where the ATP produced by PGK1 augments HSP90's ATPase-dependent chaperone activity, stabilizing client proteins and conferring resistance to cellular stresses such as oxidative damage or proteotoxic conditions. Additionally, PGK1 forms transient complexes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as part of glycolytic metabolons, enabling efficient channeling of intermediates like 1,3-bisphosphoglycerate between enzymes to optimize flux through the lower glycolysis pathway without diffusive loss. At the transcriptional level, PGK1 expression is tightly controlled by environmental cues to match metabolic demands. Under hypoxic conditions, hypoxia-inducible factor 1 (HIF-1) binds to hypoxia-responsive elements in the PGK1 promoter, leading to rapid upregulation of PGK1 mRNA and protein levels, which sustains ATP production via glycolysis when oxidative phosphorylation is impaired. This HIF-1-mediated induction is a key adaptive mechanism in oxygen-deprived tissues, ensuring cellular survival and proliferation. PGK1 activity is also subject to allosteric feedback inhibition to prevent overproduction of glycolytic intermediates. Elevated ATP/ADP ratios, indicative of high energy status, exert product inhibition on PGK1, with ADP competitively binding to the nucleotide site and reducing the enzyme's velocity in the forward glycolytic direction; this mechanism helps maintain energetic homeostasis by slowing glycolysis when cellular ATP is abundant. In parallel, ubiquitination by the E3 ligase stress-inducible protein 1 (STUB1, also known as CHIP) targets PGK1 for proteasomal degradation, providing a degradative control that attenuates glycolytic activity during stress responses. Recent investigations as of 2025 have highlighted PGK1's role in pathological responses, particularly in acute kidney injury (AKI), where its upregulation exacerbates renal damage through interactions with pyruvate kinase M2 (PKM2), promoting ferroptosis via ALOX12 activation; inhibiting PGK1 in this context protects tubular cells and mitigates injury severity in preclinical models.^[47]

Isoforms and Genetics

Human Isozymes

In humans, two isozymes of phosphoglycerate kinase exist: PGK1 and PGK2. PGK1, encoded by the X-linked gene PGK1 at locus Xq13.3, is ubiquitously expressed across all somatic tissues and plays a central role in glycolysis to support basal metabolic energy production. In contrast, PGK2 is encoded by the autosomal gene PGK2 at chromosome 3q13.31 and exhibits testis-specific expression, initiating during meiosis in spermatocytes and peaking in postmeiotic spermatids, where it accounts for approximately 80% of testicular PGK activity. These distinct expression patterns reflect their specialized functions: PGK1 maintains glycolytic flux in diverse cell types, while PGK2 supports localized ATP generation critical for spermatogenic cells. Functionally, PGK1 ensures efficient ATP synthesis under varied cellular conditions, whereas PGK2 is adapted for the unique energy demands of spermatozoa. In PGK2-null mice, spermatogenesis completes normally with preserved sperm counts and ultrastructure, but males are infertile due to severely impaired sperm motility and reduced ATP levels (approximately 23% of wild-type), highlighting PGK2's essential role in flagellar function and male fertility. No such global knockout models exist for PGK1, underscoring its indispensability for embryonic viability and broad metabolic homeostasis. The isozymes share 88% amino acid sequence identity, arising from PGK2 as an intronless retrogene derived from PGK1, but diverge in key regions that confer specialization. PGK2 features distinct amino acid substitutions clustered in its C-terminal domain, enabling targeted localization to the fibrous sheath of the sperm flagellum alongside other glycolytic enzymes. These structural adaptations facilitate compartmentalized glycolysis in the sperm principal piece. Kinetic differences further distinguish the isozymes, with PGK2 exhibiting lower substrate affinity suited to the substrate-limited environment of spermatozoa. For instance, the Km of human PGK1 for 3-phosphoglycerate is approximately 0.19 mM, supporting high-efficiency catalysis in nutrient-replete somatic cells. PGK2, by comparison, displays a higher Km for 3-phosphoglycerate (around 0.5 mM in analogous mammalian systems), optimizing activity for localized, low-concentration substrate utilization in sperm. At least 34 distinct pathogenic variants in PGK1 have been identified (as of 2023), leading to phosphoglycerate kinase 1 deficiency with variable clinical manifestations.^[48] No cases of PGK2 deficiency have been reported in humans, consistent with its restricted expression and non-essential role in somatic tissues.

Evolutionary Conservation

Phosphoglycerate kinase (PGK) is an ancient enzyme present in all three domains of life—Bacteria, Archaea, and Eukarya—indicating its origin predates the divergence of these lineages and traces back to the last universal common ancestor (LUCA).^[1] This ubiquity underscores its essential role in glycolysis, a pathway conserved across cellular life. For instance, the bacterial PGK from Escherichia coli, comprising 387 amino acids, exhibits approximately 50% sequence identity with its human counterpart, highlighting the remarkable structural preservation despite billions of years of evolution. In eukaryotes, evolutionary adaptations have expanded PGK diversity through gene duplication events, giving rise to multiple isoforms tailored for specific cellular compartments. These duplications, observed in organisms such as kinetoplastid protists and plants, enable functions like cytosolic glycolysis and plastidial or glycosomal metabolism. Additionally, some protists feature isoforms with mitochondrial targeting signals, such as in Trypanosoma vivax; however, PGK in most trypanosomes (e.g., T. brucei and T. cruzi) is primarily glycosomal—a feature absent in prokaryotes.^[1]^[11] Phylogenetic analyses confirm the deep conservation of PGK's catalytic machinery, with key residues such as Lys219 remaining invariant across all domains since LUCA, ensuring efficient phosphoryl transfer in the glycolytic reaction. This residue, along with others like Asp375 and His167 (in human numbering), forms the active site and is preserved in sequences from bacteria, archaea, and eukaryotes, reflecting strong selective pressure on core function.^[49] Functional divergence has emerged along evolutionary lines, with prokaryotic PGKs primarily dedicated to glycolysis without evidence of moonlighting activities, whereas eukaryotic variants have gained additional roles. In eukaryotes, PGK exhibits disulfide reductase activity, particularly in secreted forms that reduce plasminogen to promote angiogenesis in pathological contexts like cancer. This moonlighting function, reliant on conserved cysteine residues, is not reported in prokaryotes, suggesting an adaptation to multicellular complexity.^[1]^[19] Recent investigations into PGK homologs from extremophiles have revealed adaptations for harsh environments, such as thermostable mutations enhancing protein stability. For example, the crystal structure of PGK from the hyperthermophilic bacterium Thermotoga maritima (determined in 1997) shows a closed conformation with features contributing to thermal stability at high temperatures. Similar thermostability is noted in PGK from Thermus thermophilus, attributed to rigid domain interfaces.

Clinical and Therapeutic Relevance

Genetic Deficiencies

Phosphoglycerate kinase deficiency is an X-linked recessive disorder caused by mutations in the PGK1 gene on chromosome Xq13.3, primarily affecting males due to hemizygosity, though female carriers may exhibit mild symptoms in rare cases of skewed X-inactivation.^[48] Over 20 distinct pathogenic variants have been reported, encompassing missense, nonsense, and splicing mutations; for instance, the missense mutation R206P severely impairs enzyme function, reducing activity to approximately 10% of normal levels and leading to disrupted ATP production in energy-demanding tissues.^[50]^[48] These genetic alterations disrupt glycolysis, the primary ATP-generating pathway in erythrocytes and neurons, resulting in multisystem involvement.^[51] Clinical manifestations typically emerge in infancy or early childhood and vary widely in severity, often including nonspherocytic hemolytic anemia attributable to ATP depletion in red blood cells, progressive myopathy with exercise intolerance and muscle weakness, and neurological complications such as seizures, intellectual disability, developmental delay, and ataxia.^[48] Hemolytic anemia affects about 60% of patients, myopathy around 45%, and neurological issues nearly 50%, sometimes progressing to parkinsonism or retinopathy in severe cases.^[52] The ATP shortage particularly impacts erythrocytes, which lack mitochondria and rely solely on glycolysis, leading to cell fragility and hemolysis.^[51] Diagnosis is confirmed through measurement of phosphoglycerate kinase enzyme activity in erythrocytes, leukocytes, or fibroblasts, typically revealing residual activity below 10-25% of normal, combined with targeted sequencing of the PGK1 gene to identify causative mutations.^[50] The prevalence is unknown, with approximately 30-40 cases reported worldwide, though it may be underdiagnosed due to its rarity and phenotypic variability.^[53] Complete knockout of the Pgk1 gene in mice results in embryonic lethality, reflecting the enzyme's indispensable role in development, while conditional or hypomorphic models replicate human-like anemia and myopathy to study disease mechanisms.^[54] Management remains supportive, focusing on transfusions for anemia, anticonvulsants for seizures, and physical therapy for myopathy, with no curative treatment available; preclinical gene therapy approaches, including viral vector delivery of functional PGK1, were under investigation as of 2023 but have not yet reached clinical trials.^[52]

Roles in Disease and Targeting

Phosphoglycerate kinase 1 (PGK1) plays a pivotal role in cancer progression, particularly through its overexpression, which enhances the Warburg effect and promotes tumor metastasis. In hepatocellular carcinoma, PGK1 drives metastasis by facilitating metabolic reprogramming that amplifies aerobic glycolysis, a hallmark of the Warburg effect. Similarly, in breast cancer, PGK1 upregulation supports tumor growth and metastasis by sustaining glycolytic flux, while in lung adenocarcinoma, elevated PGK1 levels correlate with increased invasiveness and poor prognosis. Recent studies have identified PGK1 as a therapeutic target in various malignancies; for instance, the small-molecule inhibitor NG52 suppresses lesion growth in endometriosis models by inhibiting PGK1-mediated glycolysis and cell proliferation. NG52 has also shown potential in curbing tumor cell proliferation by disrupting PGK1 activity in other cancers, such as ovarian cancer. In kidney-related pathologies, recent 2025 studies indicate that PGK1 inhibition ameliorates acute kidney injury through inactivating the PKM2/ALOX12/ferroptosis pathway and contributes to diabetic kidney disease by facilitating NLRP3 inflammasome activation.^[47]^[55] Neurological disorders present opportunities for PGK1 activation as a protective strategy against neurodegeneration. A 2025 review highlights PGK1's therapeutic potential in conditions like Alzheimer's and Parkinson's diseases, where enhancing PGK1 activity may counteract protein aggregation and mitochondrial dysfunction.^[56] In Parkinson's models, PGK1 serves as a rate-limiting enzyme in neuronal glycolysis, and its upregulation reduces α-synuclein aggregation. Activation of PGK1 has also been shown to dissolve pathological aggregates in diverse neurodegenerative contexts by boosting ATP production and proteasomal degradation. In infectious diseases, PGK1 emerges as a promising drug target. Computational studies in 2025 designed inhibitors targeting phosphoglycerate kinase in Treponema pallidum, the causative agent of neurosyphilis, demonstrating enhanced binding affinity and reduced toxicity for potential therapeutic intervention.^[57] For viral infections, PGK1 facilitates productive replication of bovine herpesvirus 1 by stimulating β-catenin-dependent transcription, suggesting that PGK1 modulation could inhibit viral propagation.^[58] Therapeutic strategies targeting PGK1 include small-molecule inhibitors for oncology applications, such as NG52, which blocks glycolysis in cancer cells and reverses epithelial-mesenchymal transition. While activators for PGK1 deficiencies are under exploration, with calls for novel compounds to address hemolytic anemia, PGK1's role in activating certain prodrugs remains an area of interest, though specific HIV-related applications lack direct validation in recent reports. Within the tumor microenvironment, secreted PGK1 influences angiogenesis, with its expression linked to vascular remodeling; however, in prostate cancer, elevated PGK1 generates angiostatin, thereby inhibiting angiogenesis and tumor progression.^[59]