PKLR

Detailed Information

The PKLR gene is located on the long arm of human chromosome 1 at 1q21, spanning approximately 9.5 kb and containing 12 exons. It encodes the 574-amino-acid pyruvate kinase (PK) isoenzymes. Through differential promoter usage and alternative splicing, PKLR produces two major isoforms: PKL, predominantly expressed in the liver, and PKR, specific to red blood cells. Although derived from the same gene, these isoforms exhibit distinct regulatory and functional characteristics. PKL is mainly expressed in the liver and kidney, playing a key role in balancing glycolysis and gluconeogenesis, whereas PKR is restricted to mature erythrocytes, maintaining energy homeostasis. Structurally, pyruvate kinase functions as a homotetramer, with each subunit containing an ADP-binding site, a substrate-binding pocket, and allosteric regulatory sites, which collectively control enzymatic activity.

Biochemical Function

PKLR-encoded pyruvate kinase catalyzes the final step of glycolysis, converting phosphoenolpyruvate (PEP) and ADP into pyruvate and ATP. This reaction represents both the rate-limiting step of glycolysis and a critical node for cellular energy production. In the liver, PKL activity is tightly regulated by metabolic signals: insulin activates the transcription factor ChREBP to upregulate PKLR expression, enhancing glucose utilization and lipid synthesis, whereas glucagon suppresses PKLR expression during fasting to activate gluconeogenesis, maintaining glucose homeostasis. This dynamic regulation allows the liver to switch efficiently between anabolic and catabolic states, acting as a central regulator of systemic energy balance.

Figure 1. Hepatic PKLR inhibition suppresses key metabolic pathways, reducing glycolysis, de novo lipogenesis, and cholesterol synthesis, which correlates with lower triglyceride accumulation and improved liver metabolic balance. (Yuan M, et al., 2025)

Genetic Mutations and Hemolytic Anemia

Mutations in PKLR are the primary molecular basis of pyruvate kinase deficiency (PKD), an autosomal recessive disorder, and the second most common red blood cell enzyme deficiency after G6PD deficiency. Over 260 PKLR mutations have been identified worldwide, with missense mutations accounting for approximately 70%, followed by deletions/insertions and splice site mutations. The prevalence of specific mutations varies by population: c.1529G>A (p.Arg510Gln) is common in European and American populations; c.1456C>T (p.Arg486Trp) in Southern Europe; and c.1468C>T (p.Arg490Trp) in Asian populations. These mutations impair enzyme function through diverse mechanisms: some directly disrupt the active site, reducing substrate affinity; others alter local hydrophobicity, affecting potassium-binding sites and increasing Km; while a few mutations at the C-terminal region destabilize the tetrameric structure, collectively causing ATP deficiency and 2,3-DPG accumulation in erythrocytes. ATP depletion weakens membrane stability, and elevated 2,3-DPG decreases hemoglobin oxygen affinity, leading to chronic nonspherocytic hemolytic anemia (CNSHA).

Clinical severity in PKD is highly heterogeneous. Mild cases may be asymptomatic or exhibit minor anemia, whereas severe cases manifest in the neonatal period with hyperbilirubinemia and life-threatening anemia requiring long-term transfusions. The genotype strongly correlates with phenotype: missense mutations often retain partial enzyme activity and produce milder anemia, whereas truncating or nonsense mutations lead to near-complete loss of function. Approximately 15–20% of patients may not be diagnosed via standard enzyme activity assays due to compensatory reticulocytosis, making next-generation sequencing of PKLR the diagnostic gold standard, particularly in compound heterozygotes.

Hepatic Metabolism and Disease Associations

PKLR plays a central role in hepatic metabolic homeostasis beyond glycolysis. Systems biology analyses reveal that PKLR forms co-expression networks with key lipid metabolism genes, such as Thrsp and FASN, regulating triglyceride synthesis and mitochondrial function. Liver-specific PKLR overexpression in male mice leads to steatosis, insulin resistance, and hypercholesterolemia, associated with increased mitochondrial pyruvate flux, enhanced citrate synthesis, and accelerated de novo lipogenesis. Notably, PKLR-mediated lipid regulation shows sexual dimorphism, with higher expression in males correlating with hepatic triglyceride content, consistent with the higher prevalence and severity of MASLD in men. Mechanistically, PKLR overexpression increases pyruvate-to-acetyl-CoA flux, supplying substrates for fatty acid synthesis, and disrupts mitochondrial respiration, generating ROS and oxidative stress. PKLR also forms a positive feedback loop with the lipogenic transcription factor ChREBP, amplifying hepatic lipid accumulation. Targeted knockdown of hepatic PKLR in high-fat/high-sucrose-fed mice reduces triglycerides, plasma insulin, and cholesterol levels while improving insulin sensitivity, mediated by downregulation of DNL genes and restoration of mitochondrial function. Pharmacologically, GKAs may upregulate PKLR and exacerbate steatosis, whereas metformin inhibits PKLR induction and improves hepatic insulin sensitivity.

Role in Tumor Metabolic Reprogramming

PKLR is increasingly recognized for its role in tumor metabolism, particularly in hepatocellular carcinoma (HCC). Approximately 60–70% of HCC samples show elevated PKLR expression, which correlates negatively with tumor differentiation and patient prognosis. PKLR promotes HCC progression through two mechanisms: first, as a glycolytic enzyme, it enhances the Warburg effect, providing biosynthetic precursors for rapid proliferation; second, it regulates mitochondrial pyruvate flux, affecting citrate synthesis and NADPH production, supporting lipid synthesis and antioxidant defenses. PKLR may also contribute to immune evasion: loss of FBP1 reduces PKLR in extracellular vesicles, impairing NK cell activity. PKLR expression patterns may aid in distinguishing HCC from intrahepatic cholangiocarcinoma, as PKLR-related metabolic pathways are more active in HCC. In prostate cancer, PKLR contributes to neuroendocrine differentiation after androgen deprivation therapy, activating the CHRM4-AKT-MYCN axis, enhancing tumor aggressiveness and therapy resistance. Combined inhibition of PKLR and CHRM4 delays CRPC progression, offering a novel therapeutic strategy.

Therapeutic Target Potential and Challenges

Given its central role in multiple diseases, PKLR represents a promising therapeutic target. Gene therapy using lentiviral delivery of normal PKLR to autologous hematopoietic stem cells has corrected anemia in animal models and early-phase clinical trials, reducing transfusion requirements. Small-molecule allosteric activators, such as Mitapivat, stabilize PKR tetramers and enhance enzymatic activity, with clinical trials showing significant hemoglobin improvements in PKD patients. In metabolic liver disease and HCC, PKLR inhibitors face challenges in selectively suppressing pathological activity while preserving normal metabolism. Computational drug repositioning identified compounds like BX-912 and JNK-IN-5A as potential PKLR inhibitors, but tissue selectivity remains critical. Liver-targeted GalNAc-siRNA delivery can achieve up to 80% hepatic knockdown of PKLR without affecting erythrocyte PKR. Key challenges for future PKLR-targeted therapy include achieving tissue-specific modulation, overcoming metabolic compensation, and defining therapeutic windows. Advances in CRISPR-Cas9 gene editing, single-cell metabolomics, and AI-assisted drug design are paving the way for precise, disease-specific PKLR interventions, positioning PKLR as a hub connecting core metabolism with major human diseases and opening avenues for treating hemolytic anemia, metabolic liver disease, and cancer.