Chapter 17: Pyruvate Kinase Deficiency & Disorders of Glycolysis – Key Vocabulary

Dependence of Mature Erythrocytes on Glycolysis

• Mature red cells lack nucleus, mitochondria, ribosomes, other organelles → no DNA/RNA replication, no oxidative phosphorylation.
• Energy supplied almost exclusively by Embden–Meyerhof (glycolytic) pathway.
• ATP crucial for:
  • \mathrm{Na^+/K^+}‐ATPase (cation gradient, cell volume)
  • Ca^{2+} extrusion, maintenance of deformability
  • Phospholipid translocation (membrane asymmetry)
• 2,3-Bisphosphoglycerate (2,3-BPG) synthesized via Rapoport–Luebering shunt regulates Hb–O$_2$ affinity.
• Any block in glycolysis → ↓ATP ± altered 2,3-BPG → membrane rigidity, cation loss, premature splenic/hepatic sequestration → congenital nonspherocytic hemolytic anemias (CNSHAs).

Clinical Red Flags Suggesting Glycolytic Enzymopathy (CNSHA)

• Chronic hemolysis with:
  • Absence of spherocytes on smear
  • Normal osmotic fragility of fresh cells
  • Recessive inheritance (except X-linked PGK, dominant ADA overproduction)
  • Partial/variable benefit from splenectomy
• Neonatal jaundice, gall-stones, reticulocytosis common.
• Basophilic stippling → think pyrimidine-5′-nucleotidase (P-5′-N) deficiency.

Laboratory/Diagnostic Strategy

1. Exclude immune hemolysis, membrane defects, hemoglobinopathies.
2. Quantify suspect enzyme (beware leukocyte contamination, recent transfusion, reticulocyte surge, cell-age distribution).
3. DNA mutation analysis increasingly available.
4. Rapid screens:
   • PK, TPI, GPI spot assays possible in many labs.
   • Metabolite profiling: ↑2,3-BPG (PK, PGK), ↓2,3-BPG (HK), ↑ pyrimidine nucleotides (P-5′-N), ↓ATP (ADA over-production) etc.

Key in-vitro parameters to characterize mutant enzymes

• V_{max}: maximal velocity with saturating substrate.
• Km: substrate conc. at \frac{1}{2}V{max}; catalytic efficiency.
• pH optimum, heat stability, electrophoretic mobility, immunologic specific activity.

Hexokinase (HK) Deficiency

Clinical

• ~24 reported patients.
• Spectrum: asymptomatic → severe transfusion-dependent anemia, neonatal hyperbilirubinemia, hydrops fetalis; gall‐stones common.
• No hemolytic crises.

Biochemistry

• HK is rate-limiting, lowest-activity glycolytic enzyme.
• Normal red cells possess two isoforms from one gene (HK1): HK-R (half-life ~10 d) & HK-1 (66 d); rapid loss during reticulocyte maturation.
• Mutant activity range 13–91 % of normal; deficiency impacts young cells more (high metabolic demand).
• Mutant cells: ↓glucose utilization at low substrate, especially in acidic hypoglycemic spleen; ↓G6P, ↓2,3-BPG → left-shifted O$_2$ curve, poor exercise tolerance; ATP variably low.

Genetics

• Autosomal recessive (HK1 on 10q22).
• >8 molecular lesions: missense, 96-bp exon 5 deletion, promoter defects; compound heterozygosity common.
• Downeast mouse model: transposon insertion → severe anemia + hepatic/renal iron overload.

Therapy

• Supportive transfusion + folate.
• Splenectomy: partial benefit.

Glucose-6-Phosphate Isomerase (GPI) Deficiency

Clinical

• >60 pts; moderate–severe hemolytic anemia ± neonatal hydrops; crises with infection/drugs.
• Sub-group with neuromuscular dysfunction (GPI ≡ neuroleukin/autocrine motility factor).
• MCV 97–139 fL; post-splenectomy dense microspherocytes.

Biochemistry

• GPI homodimer (60 kDa ×2) on chr 19.
• Elevated G6P:F6P ratio; glycolysis normal in vitro but impaired in vivo.
• Resting pentose-phosphate shunt markedly ↓; cells vulnerable to oxidant stress (Heinz bodies, ↓GSH).
• Enzyme usually thermolabile → accelerated decay in aging cells.

Genetics

• Autosomal recessive.
• 31 pathogenic mutations (Table 17-3): mostly missense; homozygous missense → variable severity; compound heterozygous missense + nonsense/splice → severe.
• Mouse models replicate anemia.

Therapy

• Splenectomy—often curative for transfusion needs but anemia persists (Hb 6.7–10 g/dL).

Phosphofructokinase (PFK) Deficiency

Phenotypes (Table 17-4)

1. Type I (Tarui disease): M-subunit absent; myopathy + hemolysis.
2. Erythroid‐only deficiencies: mild anemia ± macrocytosis.
3. Pure muscle variants.
4. Asymptomatic biochemical variants (unstable L or M forms).

Clinical Hallmarks

• Exercise intolerance, cramps, myoglobinuria, hyperuricemia.
• Can be fatal in infancy or subclinical till old age.

Biochemistry

• Active PFK = tetramer; erythrocyte mix ≈50 % M‐subunit.
• M-subunit loss → unstable L$_4$ tetramers sensitive to ATP inhibition.
• Dogs: episodic alkalosis-induced hemolysis due to low 2,3-BPG.

Genetics

• PFKM gene on 12q; >15 mutations (8 missense, 5 splice, etc.).
• L-subunit gene on 21q (trisomy 21 → ↑PFK activity).

Therapy

• Avoid strenuous exercise; transfuse + folate for anemia; splenectomy rarely needed.

Aldolase A Deficiency

Cases (6 total)

• Triad: hemolytic anemia ± myopathy ± mental retardation.
• Japanese Asp128→Gly mutant & German Glu206→Lys mutant: severe enzyme thermolability.
• Sicilian compound heterozygote: premature stop + Cys338→Tyr → fatal rhabdomyolysis in fever.

Gene/Protein

• Aldolase A in erythrocyte/muscle; tetramer 40 kDa subunits.
• Missense mutations disrupt subunit interface → heat-labile.

Triose Phosphate Isomerase (TPI) Deficiency

Clinical

• ≈50 pts; infancy onset anemia + progressive neuro-degeneration (spasticity → hypotonia), recurrent infections; death <5 yr, but rare long survivors.
• Reticulocytes up to 50 %; macrocytosis.

Biochemistry

• TPI dimer 26.8 kDa; gene 12p13.
• In vitro activity 2–35 % but still >>HK; DHAP accumulates; ATP low; reliance on pentose phosphate shunt.
• Enzyme often heat-labile (classic Glu104→Asp founder mutation).

Genetics

• 14 mutations: majority Glu104→Asp; others Phe240→Leu + nonsense; compound heterozygosity common.
• Null allele homozygosity lethal in mouse embryos; Asp49→Gly knock-in mice mimic human anemia.

Therapy

• Supportive transfusion, folate; splenectomy ineffective.
• Experimental enzyme-replacement/gene therapy under study.

Glyceraldehyde-3-Phosphate Dehydrogenase (G3PD) Deficiency

• Heterozygotes with 50 % activity generally asymptomatic.
• Combined with hereditary spherocytosis → no added severity.
• Causative link with hemolysis uncertain.

Phosphoglycerate Kinase (PGK) Deficiency (X-linked)

Clinical Spectra (Table 17-6)

• Hemizygous males: (1) hemolytic anemia ± neurological disease (seizures, movement disorders) ± myopathy; (2) isolated myopathy; (3) asymptomatic (PGK München).
• Heterozygous females: mild/no anemia due to lyonisation.

Biochemistry

• Monomer 417 aa; hinge between N- and C-domains closes to bring ADP + 1,3!\text{-}BPG \rightarrow ATP + 3\text{-}PG.
• Mutants: ↓stability, altered K_m, ↓binding; cells accumulate 2,3-BPG (×2–3) and upstream metabolites; ATP near-normal.

Genetics

• 17 pathogenic mutations: 14 missense, 1 single aa deletion, 1 cryptic splice insertion (+10 aa), 1 4-aa truncation; cluster in C-domain active site (see Fig 17-4).
• Second functional gene PGK 2 on chr 19 (sperm-specific).

Therapy

• Splenectomy often reduces transfusion need; no cure.

2,3-Bisphosphoglycerate Mutase (BPGM) Deficiency

• Complete loss → 2,3-BPG nearly zero → left-shifted Hb–O$_2$ curve → compensatory polycythemia; no hemolysis.
• Compound heterozygotes (Creteil I Arg89→Cys + frameshift) best studied.
• Homozygous Arg62→Gln mutation with concomitant G6PD deficiency: no hemolysis.

Monophosphoglycerate Mutase (MPGM) Deficiency

• One spherocytosis patient homozygous Met230→Ile in MPGM-BB isozyme (50 % activity) → subtle contributor to hemolysis.

Enolase Deficiency

• Two pedigrees: severe neonatal crisis vs clinically silent.
• Triggered hemolysis by nitrofurantoin in one adult.
• Autosomal dominant partial variants demonstrated.

Pyruvate Kinase (PK) Deficiency – Most Common Glycolytic CNSHA

Clinical Spectrum

• Incidence ≈ 5.1 \times 10^{-5} in Whites.
• Neonatal jaundice, hydrops fetalis, lifelong anemia (mild to transfusion-dependent).
• Splenomegaly; paradoxical ↑reticulocytosis post-splenectomy; pigment gallstones; iron overload (low hepcidin).
• Chronic leg ulcers reported; crises rare (aplastic with infection or pregnancy).

Biochemistry

• Active tetramer; R- and L-subunits from PKLR (chr 1q21); M1/M2 from PKM (15q22).
• R-type longer by 31 aa (promoter choice).
• Allosteric regulators: K^+, Mg^{2+}, FDP (activator), ATP (inhibitor K_i\approx3.5\times10^{-4} M).
• Pathophysiology cascade:
  • ↓PK → ↑PEP ↑2,3-BPG (×3) ↓ATP.
  • Reticulocytes depend on mitochondria; in hypoxic spleen oxidative phosphorylation stops → ATP collapse → K^+ leak, water loss → rigid echinocytes (“desicytes”) → splenic/liver removal.
  • Mature cells survive nearly normally if not sequestered.
• Autohemolysis test: hemolysis >25 % after 48 h saline incub; glucose accentuates lysis if retics>25 % (Crabtree effect).

Genetics

• Autosomal recessive; >190 PKLR mutations known.
  • Types: missense (~70 %), splice, nonsense, promoter, small & large deletions/insertions.
  • Common: G1529A\,(Arg510\toGln) (N Europe) – normal kinetics but unstable; C1456T\,(Arg486\toTrp) (Mediterranean); C1468T (Asian); C1151T (Japanese).
• Compound heterozygosity frequent; homozygous null → intrauterine death/neonatal lethality.
• True heterozygotes clinically silent (PK≈50 %).
• Basenji dog, beagle, cat, mouse models: frameshift or missense in R-PK ⇒ anemia.
• Possible malaria protection under investigation (↓invasion, ↑macrophage clearance when ATP↓).

Therapy

• Splenectomy: often raises Hb 1–3 g/dL, reduces transfusion; unpredictable in mild cases.
• Transfusion + folate; monitor iron, chelate if ferritin high.
• Avoid high-dose salicylates (inhibit OXPHOS).
• Allogeneic bone marrow cure in dog/mouse and 1 human; gene therapy trials emerging; small-molecule activator AG-348 normalizes metabolism in vitro (phase study).

Lactate Dehydrogenase (LDH) Deficiency

• LDH-H or LDH-M subunit absence in humans → myopathy ± rash but no hemolysis.
• LDH-M mutant mice (<10 % activity) → severe anemia.

Disorders of Erythrocyte Nucleotide Metabolism

Pyrimidine-5′-Nucleotidase (P-5′-N) Deficiency

• Most common nucleotide enzymopathy (~100 pts).
• Moderate chronic anemia, striking basophilic stippling (up to 5 % cells), splenomegaly, iron overload; developmental delay possible.
• Biochem:
  • CMP-responsive isozyme absent; dTMP isozyme intact.
  • Accumulation of CMP, UMP, CDP-choline, CMP etc. (1.3–5× normal).
  • ↓pH (0.1–0.2), ↑GSH, secondary ↓PRPP synthetase, impaired pentose shunt.
• Gene on 7p; 27 mutations (missense, nonsense, splice, deletions). Homozygous or compound heterozygous.
• Lead poisoning produces acquired P-5′-N inhibition → similar picture.
• Splenectomy: little benefit; treat lead toxicity specifically.

Adenylate Kinase 1 (AK) Deficiency (rare)

• Autosomal recessive; >10 patients.
• Mild–severe hemolysis; some with mental retardation.
• AK1 gene on 9q34; missense, nonsense, frameshift mutations; residual activity may come from other tissue isozymes.
• Splenectomy benefited 5/6 Arab cases.

Adenosine Deaminase (ADA) Overproduction

• Autosomal dominant; 45–110× ↑ADA activity; anemia + very low adenine nucleotides.
• Mechanism: massive over-expression of normal ADA mRNA (unknown promoter defect).

Acquired Glycolytic Lesions

• Hypophosphatemia (Pi <0.3 mg/dL): ↓glycolysis → ↓ATP & 2,3-BPG → hemolytic microspherocytosis; causes—refeeding, TPN, phosphate binders, DKA, malabsorption, alcoholism.
• Hyperphosphatemia (uremia): ↑ATP (70 %), mild ↑2,3-BPG.
• Severe Mg^{2+} deficiency: experimental rat model shows similar ATP/2,3-BPG drop and rigidity.
• Iron deficiency: unstable ATP on incubation, ↑autohemolysis; suggests metabolic contribution to shortened survival.
• Hemopoietic malignancies / dyserythropoiesis: foetal enzyme profile, post-translational modification, or synthesis failure leads to complex acquired enzymopathies useful in diagnosis.

Summary of Key Metabolite Patterns

• HK ↓ → ↓G6P, ↓2,3-BPG, ↑O$_2$ affinity.
• GPI ↓ → normal/high 2,3-BPG; impaired shunt.
• PFK ↓ → ↓ATP, ↓2,3-BPG during rest; exercise may normalize intermediates.
• PK ↓ → ↑2,3-BPG, ↑triose-P, ↓ATP.
• PGK ↓ → ↑2,3-BPG, normal ATP.
• BPGM ↓ → near-zero 2,3-BPG → polycythemia.
• P-5′-N ↓ → high pyrimidines, ↑GSH, normal/low adenine nucleotides.
• ADA ↑ → very low ATP, high inosine, uric acid.

Core Equations (Embden-Meyerhof highlights)

• HK: \text{Glucose} + ATP \xrightarrow{HK} G6P + ADP
• PFK: F6P + ATP \xrightarrow{PFK} F!1,6P + ADP
• Aldolase: F!1,6P \rightarrow G3P + DHAP
• TPI: DHAP \rightleftarrows G3P
• G3PD: G3P + NAD^{+} + P_i \rightarrow 1,3\text{-}BPG + NADH
• PGK: 1,3\text{-}BPG + ADP \rightarrow 3PG + ATP
• Enolase: 2PG \rightarrow PEP + H_2O
• PK: PEP + ADP \rightarrow \text{Pyruvate} + ATP
• BPGM: 1,3\text{-}BPG \rightleftarrows 2,3\text{-}BPG (mutase/phosphatase activities)

High-Yield Connections & Implications

• Elevated 2,3-BPG in PK/PGK deficiency offsets anemia by delivering more O$_2$.
• Low 2,3-BPG in HK/PFK deficiency → tissue hypoxia despite mild anemia.
• Thermolability of mutant enzymes explains fever-triggered crises (aldolase, PGK, dog PFK).
• Band 3 docking of glycolytic enzymes (HK, G3PD, PFK) modulates flux; mutation may disrupt membrane binding undetected in soluble assays.
• Splenectomy helps when splenic environment (acidic, hypoglycemic, hypoxic) exacerbates metabolic weakness (HK, PK, PGK, GPI).
• Mouse/dog/cat natural mutants invaluable for gene-therapy, BMT, small-molecule activator trials.

Practical Study Tips

• Memorize which step each enzyme catalyzes + expected metabolite/oxygen–affinity shift when deficient.
• Know inheritance patterns: autosomal recessive (most); X-linked (PGK); dominant (ADA over-production); note Downeast HK mouse.
• Splenectomy helpful: PK, HK, GPI, PGK (variable). Useless: TPI, P-5′-N.
• Drug sensitivities: nitrofurantoin (enolase), high-dose salicylate (PK +), oxidative drugs (G6PD + select glycolytic defects).
• Distinguish congenital P-5′-N deficiency from lead poisoning (acquired enzymopathy).

End‐of-Section Quick Table (Mnemonic)

*Low at rest; can rise after intense exercise.