Enzymology & Metabolism

1. What this chapter covers, and how NEET PG actually tests it

Metabolism is the topic students most often try to memorise and most often fail to retain.

The reason is that the pathways are learned as sequences of intermediates rather than as controlled processes. Almost no NEET PG question asks for an intermediate. They ask which enzyme is rate-limiting, what regulates it, and what happens when it fails.

A pathway is therefore worth learning as three things: its rate-limiting enzyme, that enzyme's regulators, and the clinical consequence of blocking it. Everything between those points is scaffolding.

Enzyme kinetics is tested even more narrowly. Three types of inhibition exist, and questions turn entirely on what each does to Km and Vmax.

This chapter covers enzyme kinetics and inhibition, glucose oxidation from glycolysis to the citric acid cycle, oxidative phosphorylation and its poisons, and the fuel switch between fed and fasting states.

2. Enzyme kinetics

2.1 Km and Vmax mean two different things

The Michaelis-Menten equation relates reaction velocity to substrate concentration:

$v=\frac{V_{max}[S]}{K_m+[S]}$

Vmax is a capacity term — the maximum rate when the enzyme is saturated. It depends on how much functional enzyme is present.

Km is an affinity term — the substrate concentration at which velocity is half of Vmax.

A low Km means high affinity, because the enzyme reaches half its maximum rate at a low substrate concentration. The inverse relationship is the point most often reversed under pressure.

The Lineweaver-Burk plot linearises this by plotting reciprocals, giving a y-intercept of 1/Vmax and an x-intercept of −1/Km.

2.2 Three inhibition types, distinguished by two numbers

Each row follows logically from where the inhibitor binds.

A competitive inhibitor occupies the active site, so more substrate outcompetes it and the maximum rate is still achievable — only more substrate is needed, which is exactly what a raised Km describes.

A non-competitive inhibitor binds elsewhere and disables the enzyme regardless of substrate, so it effectively removes enzyme molecules from the pool. Capacity falls while the affinity of the remaining enzyme is unchanged.

An uncompetitive inhibitor binds only after substrate has bound, so it removes enzyme-substrate complex from the reaction. That pulls the binding equilibrium toward more complex formation, which appears as increased apparent affinity — hence both values fall together.

Methanol poisoning treated with fomepizole is the standard clinical example of competitive inhibition, since fomepizole competes for alcohol dehydrogenase.

3. Glucose oxidation

3.1 Two enzymes for one reaction, and why the difference matters

Glucose entering a cell is phosphorylated, but two different enzymes do this in different tissues.

The high Km of glucokinase is the whole point rather than a deficiency.

A low-affinity enzyme only works appreciably when glucose is abundant, so the liver takes up glucose after a meal and leaves it alone during fasting, when other tissues need it more.

In the beta cell the same property makes glucokinase a glucose sensor, since its activity tracks blood glucose across the physiological range instead of saturating.

That is why a glucokinase mutation causes a form of maturity-onset diabetes of the young — the sensor is miscalibrated rather than the insulin machinery being broken.

3.2 Glycolysis and its single control point

Phosphofructokinase-1 is the rate-limiting enzyme of glycolysis, and essentially all regulation acts there.

It is activated by AMP — a signal of low energy — and inhibited by ATP and citrate, both signals of plenty.

The most important activator is fructose-2,6-bisphosphate, which is not a pathway intermediate at all but a dedicated regulatory molecule.

It is made and destroyed by a single bifunctional enzyme, phosphofructokinase-2, whose activity is switched by phosphorylation.

Insulin causes dephosphorylation, favouring synthesis of fructose-2,6-bisphosphate, so glycolysis runs. Glucagon causes phosphorylation through protein kinase A, favouring its breakdown, so glycolysis stops and gluconeogenesis proceeds.

This is the central hepatic fuel switch, and it is worth knowing in preference to almost anything else in the pathway, because it simultaneously explains both directions.

Two steps generate ATP by substrate-level phosphorylation, at phosphoglycerate kinase and pyruvate kinase, and these are the only ATP-producing steps available without oxygen.

Red cells depend on this entirely, having no mitochondria, which is why they are uniquely vulnerable to glycolytic enzyme defects such as pyruvate kinase deficiency.

3.3 Pyruvate dehydrogenase and its five cofactors

Pyruvate dehydrogenase converts pyruvate to acetyl-CoA, and the reaction is irreversible.

That irreversibility has a major consequence, developed further below: carbon that becomes acetyl-CoA can never return to glucose.

The complex requires five cofactors, four of them vitamin-derived: thiamine pyrophosphate, lipoic acid, coenzyme A, FAD and NAD.

Thiamine deficiency therefore blocks this step, and pyruvate accumulates and is diverted to lactate.

That is why thiamine deficiency produces a lactic acidosis alongside its neurological features, and why the same deficiency also impairs alpha-ketoglutarate dehydrogenase, which uses the identical cofactor set.

Arsenic poisoning inhibits lipoic acid, producing a comparable block.

3.4 The citric acid cycle

Isocitrate dehydrogenase is the rate-limiting enzyme, activated by ADP and inhibited by ATP and NADH.

Each turn of the cycle, from one acetyl-CoA, yields three NADH, one FADH2 and one GTP.

The cycle is amphibolic rather than purely catabolic — its intermediates are drawn off for biosynthesis, which is why they must be replenished by anaplerotic reactions such as pyruvate carboxylase.

4. Oxidative phosphorylation

4.1 The chain and where each poison acts

Electrons pass along four complexes, pumping protons and creating the gradient that drives ATP synthase.

Complex II is worth noting separately, because it is the only citric acid cycle enzyme embedded in the inner mitochondrial membrane, which is how FADH2 delivers its electrons directly into the chain.

Because complex II bypasses complex I, FADH2 yields less ATP than NADH — fewer protons are pumped.

4.2 Inhibitors versus uncouplers

This distinction generates a reliable question, and the two behave oppositely in one specific respect.

An inhibitor blocks electron flow, so proton pumping stops, the gradient collapses and ATP synthesis fails. Oxygen consumption falls, because electrons no longer reach oxygen.

An uncoupler leaves electron transport intact but makes the inner membrane leaky to protons, so the gradient dissipates without passing through ATP synthase.

ATP synthesis fails here too, but the chain runs faster than ever because the gradient it is fighting against has disappeared.

So oxygen consumption rises, and the energy emerges as heat.

That is the mechanism of 2,4-dinitrophenol, of salicylate at toxic doses, and of the physiological uncoupling protein thermogenin in brown adipose tissue, which generates neonatal heat.

Increased oxygen consumption with hyperthermia and no ATP is therefore the uncoupler signature, and it distinguishes salicylate poisoning from a simple metabolic acidosis.

5. Fuel switching between fed and fasting states

5.1 Gluconeogenesis bypasses three irreversible steps

Gluconeogenesis is not simply reversed glycolysis, because three glycolytic steps are irreversible and must be circumvented by four enzymes.

Pyruvate carboxylase, in mitochondria, requires biotin and is activated by acetyl-CoA.

Phosphoenolpyruvate carboxykinase follows, then fructose-1,6-bisphosphatase, the rate-limiting step, inhibited by fructose-2,6-bisphosphate and AMP.

Glucose-6-phosphatase completes the process, and is found in the endoplasmic reticulum of liver and kidney only.

That restricted distribution is why muscle cannot release glucose into the blood, however much glycogen it holds — muscle glycogen serves muscle alone.

Note that fructose-2,6-bisphosphate inhibits gluconeogenesis at the same time as it activates glycolysis, so the single hepatic switch controls both directions reciprocally.

5.2 Why fat cannot become glucose

Acetyl-CoA cannot be converted to pyruvate, because pyruvate dehydrogenase is irreversible.

Even-chain fatty acids are degraded entirely to acetyl-CoA, so their carbon cannot enter gluconeogenesis. This is why prolonged starvation eventually depletes glucose despite abundant fat.

Two exceptions are examinable. Odd-chain fatty acids yield a terminal propionyl-CoA, which becomes succinyl-CoA and is therefore gluconeogenic. And glycerol released from triglyceride enters at the triose phosphate level.

So a triglyceride molecule is partly gluconeogenic through its glycerol backbone but not through its even-chain fatty acids — a distinction stems exploit.

5.3 Fatty acid oxidation and its gate

Long-chain fatty acids cannot cross the inner mitochondrial membrane unaided, and require the carnitine shuttle.

Carnitine palmitoyltransferase 1 is the rate-limiting step, and it is inhibited by malonyl-CoA.

Malonyl-CoA is the first committed intermediate of fatty acid synthesis, so its presence signals that the cell is building fat.

The cell therefore cannot synthesise and oxidise fatty acids simultaneously, and the reciprocal control is achieved by a single molecule rather than by separate signals.

Medium-chain acyl-CoA dehydrogenase deficiency is the classic defect, producing hypoketotic hypoglycaemia during fasting — the patient cannot generate ketones because fatty acid oxidation fails, and so depletes glucose instead.

The absence of ketones in a hypoglycaemic fasting child is the diagnostic clue, since ketones would be expected.

5.4 Ketone bodies

In prolonged fasting, hepatic acetyl-CoA exceeds citric acid cycle capacity and is diverted to ketone bodies, with HMG-CoA synthase as the rate-limiting enzyme.

The three ketone bodies are acetoacetate, beta-hydroxybutyrate and acetone, the last being volatile and responsible for the breath odour.

The liver makes ketones but cannot use them, lacking the enzyme required to reactivate acetoacetate.

This is a design feature rather than a limitation — an organ that consumed its own product could not export it.

The brain, which cannot oxidise fatty acids because they do not cross the blood-brain barrier readily, adapts to using ketones over several days of fasting, sharply reducing its glucose requirement.

Red cells never adapt, because ketone oxidation is mitochondrial and they have no mitochondria, so an obligatory glucose requirement always remains.

5.5 The pentose phosphate pathway and why it is not about energy

The pentose phosphate pathway branches off glucose-6-phosphate and produces no ATP at all.

Its two products are NADPH and ribose-5-phosphate, and confusing NADPH with NADH is one of the commonest errors in the subject.

NADH feeds the electron transport chain to make ATP. NADPH is a reducing agent for biosynthesis and for defence, and the two are not interchangeable.

Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme, and it is inhibited by NADPH itself, so the pathway runs only when reducing power has been consumed.

NADPH is required for fatty acid and steroid synthesis, for the respiratory burst in neutrophils, and for regenerating reduced glutathione.

That last role is the clinically important one. Reduced glutathione neutralises peroxides, and without it the red cell membrane and haemoglobin are oxidised.

Glucose-6-phosphate dehydrogenase deficiency therefore causes haemolysis specifically under oxidative stress — infection, fava beans, or drugs such as primaquine and sulphonamides — with Heinz bodies of denatured haemoglobin and the bite cells produced when splenic macrophages remove them.

The red cell is uniquely vulnerable because it cannot synthesise new enzyme, having no nucleus, so its glucose-6-phosphate dehydrogenase activity declines with cell age and the oldest cells lyse first.

That age dependence explains why the haemolysis is self-limiting and why enzyme assays performed during an acute episode can be falsely normal.

7. Common exam traps

• Reversing the Km and affinity relationship. A low Km means high affinity, because half-maximal velocity is reached at a low substrate concentration.
• Confusing uncompetitive with non-competitive inhibition. Non-competitive lowers Vmax alone; uncompetitive lowers both values.
• Treating glucokinase's high Km as a defect. It is what allows the liver to take up glucose only postprandially and the beta cell to sense glucose.
• Forgetting that fructose-2,6-bisphosphate controls both directions. It activates glycolysis and inhibits gluconeogenesis simultaneously.
• Believing fat can be converted to glucose. Pyruvate dehydrogenase is irreversible; only odd-chain fatty acids and glycerol are gluconeogenic.
• Confusing inhibitors with uncouplers. Inhibitors reduce oxygen consumption; uncouplers increase it and generate heat.
• Expecting ketosis in every fasting hypoglycaemia. Its absence indicates a fatty acid oxidation defect.
• Assuming the liver uses the ketones it makes. It lacks the reactivating enzyme, which is what allows export.

Summary

• Vmax reflects enzyme capacity while Km reflects affinity, and a low Km means high affinity.
• Competitive inhibition raises Km with unchanged Vmax and is overcome by substrate; non-competitive lowers Vmax alone; uncompetitive lowers both.
• Hexokinase has low Km and is product-inhibited, while glucokinase has high Km, is insulin-induced and acts as the liver and beta cell glucose sensor.
• Phosphofructokinase-1 is the rate-limiting enzyme of glycolysis, activated by AMP and fructose-2,6-bisphosphate and inhibited by ATP and citrate.
• Insulin promotes and glucagon suppresses fructose-2,6-bisphosphate through the bifunctional phosphofructokinase-2, which is the central hepatic fuel switch.
• Substrate-level phosphorylation at phosphoglycerate kinase and pyruvate kinase is the only ATP source without oxygen, which is why red cells depend on glycolysis alone.
• Pyruvate dehydrogenase is irreversible and requires five cofactors, so thiamine deficiency blocks it and causes lactic acidosis.
• Isocitrate dehydrogenase is rate-limiting for the citric acid cycle, which yields three NADH, one FADH2 and one GTP per acetyl-CoA and is amphibolic.
• Electron transport inhibitors act at defined complexes, with cyanide and carbon monoxide at complex IV and oligomycin at ATP synthase.
• Complex II is the only citric acid cycle enzyme in the inner membrane, and its bypass of complex I is why FADH2 yields less ATP.
• Inhibitors reduce oxygen consumption; uncouplers increase it while producing heat instead of ATP, which is the salicylate and dinitrophenol mechanism.
• Gluconeogenesis bypasses three irreversible steps using pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase.
• Glucose-6-phosphatase is confined to liver and kidney, which is why muscle glycogen cannot raise blood glucose.
• Even-chain fatty acids cannot become glucose because pyruvate dehydrogenase is irreversible, but odd-chain fatty acids and glycerol can.
• Carnitine palmitoyltransferase 1 gates fatty acid oxidation and is inhibited by malonyl-CoA, so synthesis and oxidation cannot run together.
• Hypoketotic hypoglycaemia indicates a fatty acid oxidation defect, and the liver produces but cannot consume ketone bodies, while red cells can never use them.
• The pentose phosphate pathway makes no ATP; its products are NADPH for biosynthesis and antioxidant defence, and ribose-5-phosphate for nucleotides.
• Glucose-6-phosphate dehydrogenase deficiency causes oxidative haemolysis with Heinz bodies and bite cells, and enzyme assays during an acute episode can be falsely normal because the oldest, most deficient cells have already lysed.