STEPTRACKERBIOCHEMISTRY + HEMATOLOGY | CARBOHYDRATE METABOLISM | HIGH-YIELD REVIEW

Glycolysis

Glucose → 2 pyruvate in the cytosol of every cell: three irreversible steps, two ATP spent, four made — and a red cell that lives on it alone.

FOCUS ON
Irreversible, regulated steps: hexokinase/glucokinase, PFK-1 (rate-limiting), pyruvate kinase. Hexokinase: all tissues, low Km, inhibited by G6P. Glucokinase: liver and β cells, high Km/high Vmax, induced by insulin, held in the nucleus by GKRP. Net 2 ATP + 2 NADH per glucose. Pyruvate kinase deficiency → hemolytic anemia (↑ 2,3-BPG). Fluoride inhibits enolase; arsenate bypasses an ATP-making step.
STEP · ENZYME
REACTION
REGULATION
CLUE · DISEASE
Hexokinase
most tissues
Glucose → glucose-6-P (uses ATP; traps glucose)
Low Km (high affinity), low Vmax; inhibited by G6P
Keeps glycolysis going even at low glucose (brain, RBCs)
Glucokinase
liver, β cells
Same reaction
High Km, high Vmax; induced by insulin; inhibited by F-6-P (via GKRP); not by G6P
Glucose sensor · MODY 2 (GCK mutation, mild fasting hyperglycemia)
Phosphofructokinase-1
rate-limiting
Fructose-6-P → fructose-1,6-BP (uses ATP)
+ AMP, F-2,6-BP · − ATP, citrate
Tarui disease (PFK deficiency) — muscle cramps, hemolysis
PFK-2 / FBPase-2
Makes or breaks F-2,6-BP
Fed (insulin): PFK-2 active → ↑ F-2,6-BP → glycolysis · fasting (glucagon, PKA): FBPase-2 active → ↓ F-2,6-BP → gluconeogenesis
Same bifunctional enzyme; phosphorylation flips it
Glyceraldehyde-3-P dehydrogenase
G3P → 1,3-BPG (makes NADH)
Needs NAD⁺ (regenerated by lactate DH when no O₂)
—
Phosphoglycerate kinase
1,3-BPG → 3-PG (makes ATP)
—
Arsenate substitutes for phosphate → this ATP is lost
Bisphosphoglycerate mutase
RBCs
1,3-BPG → 2,3-BPG (Rapoport-Luebering shunt)
↑ In hypoxia, altitude, anemia
2,3-BPG shifts O₂ curve right
Enolase
2-PG → phosphoenolpyruvate
—
Fluoride inhibits (blood-glucose tubes)
Pyruvate kinase
PEP → pyruvate (makes ATP)
+ F-1,6-BP (feed-forward) · − ATP, alanine, glucagon (phosphorylation)
PK deficiency (AR): hemolytic anemia, echinocytes, ↑ 2,3-BPG
Lactate dehydrogenase
Pyruvate → lactate, regenerates NAD⁺
Active in RBCs, exercising muscle, hypoxia
Lactic acidosis in shock
Net yield
Glucose → 2 pyruvate
2 ATP + 2 NADH (anaerobic: 2 ATP total)
Aerobic total ~30–32 ATP per glucose

Mechanism — the three control points

Hexokinase / glucokinaseGlucose → G6P
→
F-6-P
→
PFK-1→ F-1,6-BP
→
G3P → 1,3-BPG → 3-PG → PEPNADH, ATP
→
Pyruvate kinase→ pyruvate, ATP

Each irreversible step needs a separate bypass enzyme for gluconeogenesis to run backward (8.04).

Hexokinase vs glucokinase

FEATURE
HEXOKINASE
GLUCOKINASE
Location
Most tissues
Liver, pancreatic β cells
Km
Low (high affinity)
High (low affinity)
Vmax
Low
High
Inhibited by
Glucose-6-P
F-6-P (via GKRP)
Induced by insulin
No
Yes

How it's tested

COMMON CONFUSIONGlucokinase's high Km lets the liver clear glucose only when levels are high, leaving glucose for the brain when levels are low.
THE TRAPRBCs depend entirely on glycolysis — pyruvate kinase deficiency gives hemolysis; ↑ 2,3-BPG improves O₂ delivery, so patients tolerate the anemia.
YOUR APPROACHFind the step → irreversible or not → regulator (AMP/ATP, F-2,6-BP, insulin/glucagon) → poison or deficiency at that step.

If you see… think…

Rate-limiting enzyme of glycolysis → PFK-1
Most potent PFK-1 activator → fructose-2,6-bisphosphate
Liver enzyme with high Km for glucose → glucokinase
Mild fasting hyperglycemia in a young family, glucose sensor gene → MODY 2 (GCK)
Hemolysis, echinocytes, ↑ 2,3-BPG → pyruvate kinase deficiency
Poison in gray-top blood tubes → fluoride (enolase)
Arsenate toxicity in glycolysis → no ATP from phosphoglycerate kinase
Enzyme regenerating NAD⁺ without oxygen → lactate dehydrogenase
Muscle cramps + hemolysis with exercise → PFK deficiency (Tarui)
Net ATP of anaerobic glycolysis → 2