enolase

**Biochemistry** - Reaction: 2-phosphoglycerate → phosphoenolpyruvate (PEP) + H2O. It is a reversible lyase (dehydratase) reaction in glycolysis and gluconeogenesis. - It is the penultimate step of glycolysis. Pyruvate kinase catalyzes the next step, PEP → pyruvate, which generates ATP by substrate-level phosphorylation. - Preceding step: phosphoglycerate mutase converts 3-phosphoglycerate to 2-phosphoglycerate. - The reaction needs Mg2+ as a cofactor. Dehydration creates a high-energy enol phosphate, so PEP has a very high phosphoryl-transfer potential. - Enolase is not a major regulatory step. It operates near equilibrium and is reversible, so it also functions in gluconeogenesis.

**Inhibition** - Fluoride inhibits enolase by forming a complex with Mg2+ and phosphate at the active site. - Clinical pearl: gray-top blood tubes contain sodium fluoride (often with potassium oxalate). Fluoride blocks glycolysis in RBCs, WBCs and platelets, which prevents falsely low glucose readings in stored blood samples. - Without fluoride, glycolysis continues in the tube and lowers the measured glucose. - Because enolase is inhibited, 2-phosphoglycerate and 3-phosphoglycerate accumulate, and there is less ATP production from downstream steps.

**Isoenzymes and clinical significance** - Isoforms: alpha (ubiquitous), beta (muscle), and gamma (neurons and neuroendocrine cells). - Neuron-specific enolase (NSE) is the gamma-gamma form. It is a tumor marker for: - Small cell lung cancer (neuroendocrine origin), used for monitoring treatment response and relapse. - Neuroblastoma, which is also followed by urinary HVA/VMA. - Other neuroendocrine tumors, such as carcinoid and medullary thyroid carcinoma (less specific). - NSE is also a prognostic marker after cardiac arrest. Persistently elevated serum levels at 48–72 hours suggest severe hypoxic-ischemic brain injury and a poor neurologic outcome. - Hemolysis falsely elevates NSE because RBCs contain enolase. - Alpha-enolase can be a surface plasminogen receptor on some cells. Anti-alpha-enolase antibodies have been described in autoimmune conditions (e.g., Hashimoto encephalopathy, RA, and some vasculitides), but this is low-yield. - Enolase is a virulence-associated protein in several microbes, where it can bind plasminogen. This is low-yield.

**Glycolysis enzyme quick review (high-yield)** - Irreversible/regulated steps: hexokinase/glucokinase, PFK-1 (rate-limiting), and pyruvate kinase. - Substrate-level phosphorylation steps: phosphoglycerate kinase (1,3-BPG → 3-PG) and pyruvate kinase (PEP → pyruvate). - Enolase is in the payoff phase, between phosphoglycerate mutase and pyruvate kinase.

**Deficiency** - Enolase deficiency is extremely rare. Beta-enolase deficiency (muscle) may cause exercise intolerance. Glycolytic enzyme defects that cause hemolytic anemia are most commonly pyruvate kinase deficiency (the high-yield one), then hexokinase and others.

**Board pearls** - Enolase = dehydration, 2-PG → PEP, inhibited by fluoride. - NSE: small cell lung cancer and neuroblastoma marker. - Gray-top tube (NaF) is used for glucose and lactate measurement.