Glycolysis & TCA Cycle

Glycolysis and the citric acid (Krebs/TCA) cycle form the central highway of carbohydrate oxidation. For NEET PG, the recurring questions are not vague concepts but specific enzymes, their regulation, energy yield, and the disease produced when one enzyme fails. Master the irreducible, the irreversible, and the inhibited — that is where the marks are.

Glycolysis: the basics

Glycolysis is the cytosolic conversion of one molecule of glucose (6C) into two molecules of pyruvate (3C). It is the only pathway that runs in all cells, occurs with or without oxygen, and is the sole energy source for cells lacking mitochondria (mature RBCs, corneal cells, renal medulla, and largely the lens).

• Location: cytoplasm (no organelle needed → why RBCs survive on it).
• Net ATP (aerobic): 2 ATP by substrate-level phosphorylation + NADH that yields more via the electron transport chain.
• Net ATP (anaerobic): only 2 ATP, with NADH reoxidised by converting pyruvate → lactate.

High-yield: The mature erythrocyte depends entirely on anaerobic glycolysis; it has no mitochondria, so it cannot use the TCA cycle or oxidative phosphorylation.

The ten steps (energy-investment vs energy-payoff)

Glycolysis has an investment phase (steps 1–5, spends 2 ATP) and a payoff phase (steps 6–10, generates 4 ATP + 2 NADH).

Glucose → G6P → F6P → F1,6-BP → (split) → G3P (×2) → 1,3-BPG → 3-PG → 2-PG → PEP → Pyruvate

Step	Reaction	Enzyme	Note
1	Glucose → G6P	Hexokinase / Glucokinase	Irreversible; uses ATP; traps glucose
2	G6P → F6P	Phosphoglucose isomerase	Reversible
3	F6P → F1,6-BP	PFK-1	Irreversible; rate-limiting; uses ATP
4	F1,6-BP → DHAP + G3P	Aldolase B	Cleavage (Aldolase B defect → hereditary fructose intolerance)
5	DHAP ⇌ G3P	Triose phosphate isomerase	Funnels both trioses into G3P
6	G3P → 1,3-BPG	G3P dehydrogenase	Produces NADH; uses inorganic Pi
7	1,3-BPG → 3-PG	Phosphoglycerate kinase	Substrate-level ATP (×2)
8	3-PG → 2-PG	Phosphoglycerate mutase
9	2-PG → PEP	Enolase	Loses water; inhibited by fluoride
10	PEP → Pyruvate	Pyruvate kinase	Irreversible; substrate-level ATP (×2)

High-yield: The three irreversible (regulated) steps are catalysed by hexokinase, PFK-1, and pyruvate kinase. PFK-1 is the committed, rate-limiting step of glycolysis.

Hexokinase vs Glucokinase — a classic comparison

Property	Hexokinase	Glucokinase
Tissue	Most tissues	Liver, pancreatic β-cells
Km for glucose	Low (~0.1 mM) → high affinity	High (~10 mM) → low affinity
Vmax	Low	High
Inhibited by G6P?	Yes (product inhibition)	No
Induced by insulin?	No	Yes
Role	Works even at low glucose	Acts when glucose is abundant (post-meal); glucose sensor of β-cell

High-yield: Glucokinase is the glucose sensor of the pancreatic β-cell. MODY type 2 is caused by a glucokinase (GCK) mutation. Hexokinase is inhibited by its own product G6P; glucokinase is not.

Regulation of glycolysis

The master regulator is PFK-1.

• Activators of PFK-1: AMP, and the most potent allosteric activator fructose-2,6-bisphosphate (F-2,6-BP).
• Inhibitors of PFK-1: ATP and citrate (signals of energy/biosynthetic sufficiency) and low pH.

F-2,6-BP is made/degraded by the bifunctional enzyme PFK-2/FBPase-2, controlled by phosphorylation:

Fed state (insulin) → dephosphorylated → PFK-2 active → ↑F-2,6-BP → glycolysis ON Fasting (glucagon → cAMP → PKA) → phosphorylated → FBPase-2 active → ↓F-2,6-BP → gluconeogenesis ON

High-yield: Fructose-2,6-bisphosphate simultaneously activates PFK-1 (glycolysis) and inhibits fructose-1,6-bisphosphatase (gluconeogenesis), preventing a futile cycle. Insulin raises it; glucagon lowers it.

Pyruvate kinase is also regulated: activated by F-1,6-BP (feed-forward) and inhibited by ATP, alanine, and glucagon-mediated phosphorylation.

Fate of pyruvate

Pyruvate sits at a major metabolic branch point:

1. Aerobic: → Acetyl-CoA (by pyruvate dehydrogenase) → enters TCA cycle.
2. Anaerobic: → Lactate (by lactate dehydrogenase, regenerating NAD⁺).
3. → Oxaloacetate (by pyruvate carboxylase) → gluconeogenesis/anaplerosis.
4. → Alanine (transamination).

Pyruvate dehydrogenase (PDH) complex

PDH is the irreversible bridge between glycolysis and the TCA cycle, located in the mitochondrial matrix. It needs five cofactors — the classic mnemonic "Tender Loving Care For Nancy":

• Thiamine pyrophosphate (B1)
• Lipoic acid
• Coenzyme A (B5/pantothenate)
• FAD (B2/riboflavin)
• NAD⁺ (B3/niacin)

High-yield: PDH deficiency causes congenital lactic acidosis and neurological deficits; it is one of the few organic causes treated partly with a ketogenic (high-fat) diet because the carbohydrate route is blocked. Arsenic inhibits lipoic acid (and PDH/α-KG dehydrogenase).

The TCA (Krebs / citric acid) cycle

The TCA cycle is the final common oxidative pathway for carbohydrates, fats, and proteins. It runs in the mitochondrial matrix (except succinate dehydrogenase, which is on the inner membrane = Complex II). Per one acetyl-CoA (2C entering), the cycle produces 3 NADH, 1 FADH₂, 1 GTP, and 2 CO₂.

Acetyl-CoA + OAA → Citrate → Isocitrate → α-KG → Succinyl-CoA → Succinate → Fumarate → Malate → OAA

Step	Reaction	Enzyme	Product
1	Acetyl-CoA + OAA → Citrate	Citrate synthase	—
2	Citrate → Isocitrate	Aconitase	(inhibited by fluoroacetate)
3	Isocitrate → α-KG	Isocitrate dehydrogenase	NADH + CO₂; rate-limiting
4	α-KG → Succinyl-CoA	α-KG dehydrogenase	NADH + CO₂; needs same 5 cofactors as PDH
5	Succinyl-CoA → Succinate	Succinate thiokinase	GTP (substrate-level)
6	Succinate → Fumarate	Succinate dehydrogenase	FADH₂; Complex II
7	Fumarate → Malate	Fumarase	—
8	Malate → OAA	Malate dehydrogenase	NADH

High-yield: Isocitrate dehydrogenase is the rate-limiting enzyme of the TCA cycle (activated by ADP/Ca²⁺, inhibited by ATP/NADH). Succinate dehydrogenase is the only TCA enzyme that is membrane-bound and the only one that is inhibited by malonate (competitive). It is also Complex II of the ETC and the only enzyme common to both the TCA cycle and the respiratory chain.

A useful mnemonic for the cycle: "Citrate Is Krebs' Starting Substrate For Making Oxaloacetate" (Citrate, Isocitrate, α-Ketoglutarate, Succinyl-CoA, Succinate, Fumarate, Malate, Oxaloacetate).

Regulation of the TCA cycle

• Activators: ADP, Ca²⁺ (during muscle contraction), NAD⁺.
• Inhibitors: ATP, NADH, succinyl-CoA (citrate synthase, ICDH, α-KG DH are control points).
• The cycle is purely aerobic in effect: although no step uses O₂ directly, it halts without oxygen because NADH/FADH₂ cannot be reoxidised.

High-yield: The TCA cycle is amphibolic — both catabolic (energy) and anabolic (supplies precursors: α-KG → glutamate, OAA → aspartate, succinyl-CoA → heme/porphyrins, citrate → fatty acids).

Energy yield (ATP accounting)

Using the modern P/O ratios (NADH = 2.5 ATP, FADH₂ = 1.5 ATP):

Source	Per glucose	ATP
Glycolysis (substrate-level)	2 ATP	2
Glycolysis NADH (2)	via shuttle	3 or 5
PDH NADH (2)	2 × 2.5	5
TCA: 6 NADH	6 × 2.5	15
TCA: 2 FADH₂	2 × 1.5	3
TCA: 2 GTP	substrate-level	2
Total (aerobic)		30–32 ATP

The variability (30 vs 32) depends on the shuttle used for cytosolic NADH:

• Malate–aspartate shuttle (liver, heart, kidney) → NADH → 2.5 ATP each → total 32.
• Glycerophosphate shuttle (muscle, brain) → FADH₂ → 1.5 ATP each → total 30.

High-yield: Older texts quote 38 ATP (using NADH=3, FADH₂=2). NEET PG now expects 30–32 ATP by current values. Anaerobic glycolysis yields only 2 ATP.

Note that the substrate-level phosphorylation steps are independent of oxygen and the electron transport chain — they generate ATP directly by transfer of a high-energy phosphate from a substrate (1,3-BPG and PEP in glycolysis; succinyl-CoA in the TCA cycle). This is why glycolysis still yields a net 2 ATP even under complete anoxia. By contrast, the bulk of cellular ATP (about 26–28 of the 30–32) comes from oxidative phosphorylation driven by the NADH and FADH₂ harvested in these pathways. A cell starved of oxygen (ischaemia) is therefore forced into anaerobic glycolysis, accumulates lactate, and develops a high anion-gap metabolic acidosis (type A lactic acidosis) — the biochemical basis of the lactate marker used in sepsis and shock.

Clinically relevant enzyme deficiencies

Enzyme defect	Disease / consequence	Key clue
Pyruvate kinase deficiency	Hereditary non-spherocytic haemolytic anaemia (2nd commonest after G6PD)	Autosomal recessive; ↑2,3-BPG → right-shifted O₂ curve (better tissue O₂ → relatively well tolerated); no Heinz bodies
Hexokinase deficiency	Haemolytic anaemia	↓2,3-BPG → left shift (worse symptoms)
PDH deficiency	Congenital lactic acidosis, neuro deficits, ↑alanine	X-linked; treat with ketogenic diet, thiamine
Aldolase B	Hereditary fructose intolerance	Hypoglycaemia after fructose/sucrose; aversion to sweets
Fumarase / SDH mutations	Hereditary leiomyomatosis, paraganglioma, pheochromocytoma	"Pseudohypoxia," ↑HIF; tumour-suppressor role

High-yield: Pyruvate kinase deficiency is the most common glycolytic enzyme defect causing haemolytic anaemia; it spares ATP-poor RBCs and causes 2,3-BPG accumulation that right-shifts the oxygen dissociation curve.

Key poisons / inhibitors (favourite MCQ)

• Fluoride → inhibits enolase (step 9) → used in grey-top blood collection tubes to halt glycolysis and preserve glucose.
• Arsenate → uncouples step 6 (arsenolysis of 1,3-BPG) → loss of ATP at step 7.
• Iodoacetate → inhibits glyceraldehyde-3-phosphate dehydrogenase.
• Fluoroacetate ("lethal synthesis") → forms fluorocitrate → inhibits aconitase (TCA).
• Malonate → competitive inhibitor of succinate dehydrogenase.
• Arsenite → inhibits lipoic acid-requiring enzymes (PDH, α-KG DH).

High-yield: Fluoride inhibits enolase — remember the grey-coloured fluoride/oxalate tube preserves blood glucose for accurate fasting sugar.

The Rapoport–Luebering shunt (RBC special)

In red cells, 1,3-BPG can be diverted to 2,3-bisphosphoglycerate (2,3-BPG) by bisphosphoglycerate mutase, bypassing ATP-generating step 7. 2,3-BPG binds deoxyhaemoglobin and right-shifts the oxygen dissociation curve, aiding tissue oxygen delivery (relevant in anaemia, high altitude). The trade-off: each molecule shunted to 2,3-BPG costs the RBC the ATP it would otherwise have made at step 7 — a deliberate sacrifice of energy for improved oxygen unloading. This explains the paradox in pyruvate kinase deficiency: the block is upstream of 2,3-BPG accumulation, so 2,3-BPG rises, the curve shifts right, and patients tolerate surprisingly low haemoglobin levels because oxygen delivery per gram is enhanced. The opposite occurs in hexokinase deficiency, where the proximal block lowers all downstream intermediates including 2,3-BPG, causing a left shift and worse symptoms for an equivalent anaemia.

The Warburg effect deserves a mention: many tumours preferentially use aerobic glycolysis (high glucose uptake with lactate production even when oxygen is plentiful), the principle exploited by FDG-PET imaging. This is increasingly tested as a link between metabolism and oncology.

Key differentials / overlaps to keep straight

• Glycolysis vs gluconeogenesis: share 7 reversible steps; differ at the 3 irreversible glycolytic steps, bypassed by 4 gluconeogenic enzymes (pyruvate carboxylase, PEPCK, F-1,6-bisphosphatase, glucose-6-phosphatase).
• PDH vs pyruvate carboxylase: PDH makes acetyl-CoA (fed); pyruvate carboxylase makes OAA (fasting) — acetyl-CoA activates pyruvate carboxylase.
• α-KG dehydrogenase vs PDH: identical 5 cofactors; both blocked by arsenite, both produce NADH + CO₂.

Recently asked / exam angle

• Which enzyme is rate-limiting in glycolysis? → PFK-1; in TCA → isocitrate dehydrogenase.
• Most potent allosteric activator of PFK-1 → fructose-2,6-bisphosphate.
• Enzyme common to TCA cycle and ETC → succinate dehydrogenase (Complex II).
• Substrate-level phosphorylation steps → phosphoglycerate kinase, pyruvate kinase (glycolysis); succinate thiokinase (TCA, gives GTP).
• Fluoride inhibits which glycolytic enzyme → enolase.
• Glucose sensor of β-cell / MODY 2 enzyme → glucokinase.
• Cofactors of PDH (5) → TPP, lipoic acid, CoA, FAD, NAD⁺.
• Net ATP anaerobic glycolysis → 2; aerobic complete oxidation → 30–32.
• Enzyme deficiency causing haemolytic anaemia with ↑2,3-BPG and right-shifted O₂ curve → pyruvate kinase.
• Only TCA enzyme bound to inner mitochondrial membrane → succinate dehydrogenase.

Rapid revision

1. PFK-1 = rate-limiting/committed step of glycolysis; activated by F-2,6-BP and AMP, inhibited by ATP and citrate.
2. Three irreversible glycolytic enzymes: hexokinase, PFK-1, pyruvate kinase.
3. Glucokinase = high Km, not inhibited by G6P, β-cell glucose sensor, mutated in MODY 2.
4. Glycolysis is cytosolic; mature RBC runs only anaerobic glycolysis → 2 ATP.
5. Fluoride inhibits enolase → grey-top tube preserves glucose.
6. PDH and α-KG dehydrogenase share 5 cofactors (TPP, lipoate, CoA, FAD, NAD⁺); inhibited by arsenite.
7. Isocitrate dehydrogenase = rate-limiting enzyme of the TCA cycle.
8. Succinate dehydrogenase = Complex II, membrane-bound, inhibited by malonate, only enzyme shared by TCA and ETC.
9. One acetyl-CoA → 3 NADH + 1 FADH₂ + 1 GTP + 2 CO₂.
10. Complete glucose oxidation → 30–32 ATP (malate-aspartate shuttle = 32; glycerophosphate = 30).
11. Pyruvate kinase deficiency = commonest glycolytic cause of haemolytic anaemia; ↑2,3-BPG, right-shifted O₂ curve.
12. TCA cycle is amphibolic: α-KG→glutamate, OAA→aspartate, succinyl-CoA→heme, citrate→fatty acids.