Enzyme Kinetics & Inhibition
Biochemistry · Enzymes · lean revision notes
Enzyme Kinetics & Inhibition
Enzyme kinetics quantifies how fast enzymes convert substrate to product and how that rate is altered by inhibitors, modulators, and conditions. For NEET PG this chapter is a perennial favourite — expect direct questions on Km/Vmax, the three classic inhibition patterns, Lineweaver-Burk shifts, allosteric regulation, and the clinical use of isoenzymes (LDH, CK, ALP). Master the patterns and the graphs, and the marks are predictable.
Basic concepts & definitions
An enzyme is a biological catalyst (almost always protein; ribozymes are RNA) that lowers the activation energy (Ea) of a reaction without altering the equilibrium constant or the ΔG of the overall reaction. It increases the rate of both forward and reverse reactions equally, simply by stabilising the transition state.
Key terms you must distinguish:
| Term | Meaning |
|---|---|
| Apoenzyme | Protein part alone (inactive) |
| Cofactor | Non-protein helper — metal ion (Zn²⁺, Mg²⁺) or organic molecule |
| Coenzyme | Organic, loosely bound cofactor (often vitamin-derived, e.g. NAD⁺, FAD, TPP) |
| Prosthetic group | Tightly/covalently bound cofactor (e.g. haem, biotin, FAD in succinate dehydrogenase) |
| Holoenzyme | Apoenzyme + cofactor = active enzyme |
| Isoenzymes | Different molecular forms catalysing the same reaction (e.g. LDH₁–₅) |
High-yield: Enzymes lower activation energy and do NOT change ΔG, Keq, or the position of equilibrium. They only make equilibrium arrive faster.
Enzyme classification (IUBMB)
Six (now seven) classes — remember "Over The Hill, Like Iron Lady":
- Oxidoreductases — redox (dehydrogenases, oxidases)
- Transferases — group transfer (kinases, transaminases)
- Hydrolases — hydrolysis (peptidases, lipases, phosphatases)
- Lyases — add/remove groups forming double bonds (aldolase, decarboxylases)
- Isomerases — rearrangements (epimerases, mutases)
- Ligases — join two molecules with ATP (carboxylases, synthetases)
- Translocases (newest class — membrane transport)
High-yield: "Synthetase" = ligase (needs ATP). "Synthase" = lyase (no ATP). This wording trap is repeatedly tested.
Michaelis-Menten kinetics
The Michaelis-Menten model assumes the simple scheme:
E + S ⇌ ES → E + P
Applying the steady-state assumption (Briggs-Haldane: [ES] is constant), we get the central equation:
v = (Vmax × [S]) / (Km + [S])
- Vmax = maximum velocity when the enzyme is fully saturated with substrate.
- Km (Michaelis constant) = the substrate concentration at which velocity is half of Vmax (v = Vmax/2).
The v vs [S] plot is a rectangular hyperbola:
- At low [S] (≪ Km): reaction is first-order (rate ∝ [S]).
- At high [S] (≫ Km): reaction is zero-order (rate independent of [S], v ≈ Vmax).
Reasoning flow: Set v = Vmax/2 in the equation → Vmax/2 = Vmax·[S]/(Km+[S]) → Km + [S] = 2[S] → Km = [S]. This proves Km equals the substrate concentration at half-maximal velocity.
What Km tells you
High-yield: Km is inversely proportional to enzyme–substrate affinity. A low Km = high affinity (enzyme reaches half-max at low [S]); a high Km = low affinity.
- Km is a constant for a given enzyme–substrate pair at fixed conditions; it is independent of enzyme concentration.
- Vmax is dependent on enzyme concentration (Vmax = kcat × [E]total).
- kcat (turnover number) = number of substrate molecules converted per enzyme per second at saturation. Carbonic anhydrase has one of the highest (~10⁶/sec).
- kcat/Km = the specificity constant, the best measure of catalytic efficiency; its upper limit (~10⁸–10⁹) is set by diffusion ("catalytically perfect" enzymes, e.g. catalase, fumarase, acetylcholinesterase).
High-yield classic example: Glucokinase (liver/β-cell, hexokinase IV) has a high Km (low affinity, ~10 mM) and is not inhibited by its product G6P, so it acts only when glucose is high (post-prandial). Hexokinase has a low Km (high affinity), is product-inhibited, and saturates at normal glucose — ensuring constant glucose uptake in other tissues.
Lineweaver-Burk (double-reciprocal) plot
Because the hyperbola makes Vmax hard to read precisely, we take reciprocals to linearise:
1/v = (Km/Vmax)(1/[S]) + 1/Vmax
This straight line gives:
| Axis feature | Value |
|---|---|
| Y-intercept | 1/Vmax |
| X-intercept | −1/Km |
| Slope | Km/Vmax |
High-yield: Reading inhibition off a Lineweaver-Burk plot is one of the most asked image-based questions in NEET PG. Memorise which intercept moves.
(Other linearisations exist — Eadie-Hofstee [v vs v/S] and Hanes-Woolf [S/v vs S] — occasionally asked, but Lineweaver-Burk dominates.)
Enzyme inhibition
Inhibition is reversible (non-covalent, washable) or irreversible (covalent, e.g. aspirin acetylating COX, organophosphates on acetylcholinesterase, DFP, penicillin on transpeptidase, allopurinol's oxypurinol on xanthine oxidase).
The three reversible patterns are the heart of this topic:
| Feature | Competitive | Non-competitive | Uncompetitive |
|---|---|---|---|
| Inhibitor binds | Active site (competes with S) | Site other than active site; binds E or ES | Only the ES complex |
| Structural resemblance to S | Yes (substrate analogue) | No | No |
| Km | ↑ (increased) | Unchanged | ↓ (decreased) |
| Vmax | Unchanged | ↓ (decreased) | ↓ (decreased) |
| Vmax/Km ratio | ↓ | ↓ | Unchanged |
| Overcome by ↑[S]? | Yes | No | No |
| Lineweaver-Burk | Lines meet on Y-axis (same 1/Vmax) | Lines meet on X-axis (same −1/Km) | Parallel lines |
High-yield memory hook:
- Competitive → Km changes, Vmax same → "Competition Keeps Vmax."
- Non-competitive → Vmax changes, Km same.
- Uncompetitive → both Km and Vmax decrease (both fall together).
Worked clinical examples
- Competitive: Methanol/ethylene glycol poisoning treated with ethanol or fomepizole (competes for alcohol dehydrogenase). Statins compete with HMG-CoA reductase. Allopurinol vs xanthine oxidase (also has an irreversible component). Dicoumarol vs vitamin K. Sulfonamides vs PABA (dihydropteroate synthase). Malonate vs succinate (succinate dehydrogenase) — the textbook example.
- Non-competitive: Heavy metals (lead, mercury) binding −SH groups; physostigmine/cyanide on certain enzymes.
- Uncompetitive (rare, hence high-yield): Lithium on inositol monophosphatase.
High-yield: In competitive inhibition, the apparent Km rises but Vmax is unchanged because flooding the system with substrate displaces the inhibitor. This is why fomepizole/ethanol works — it is out-competed once metabolised.
Allosteric / irreversible add-ons
- Suicide (mechanism-based) inhibitors: the enzyme converts the inhibitor into a reactive species that then irreversibly binds it — e.g. allopurinol → oxypurinol, 5-fluorouracil → FdUMP on thymidylate synthase, and DFMO (eflornithine) on ornithine decarboxylase.
Allosteric regulation & cooperativity
Allosteric enzymes do not obey Michaelis-Menten kinetics. They have multiple subunits and a regulatory (allosteric) site distinct from the catalytic site. Their v vs [S] plot is sigmoidal (S-shaped), reflecting cooperativity (binding of one substrate molecule alters affinity at other sites — as in haemoglobin's O₂ binding).
- Positive modulators shift the curve left (↓ apparent Km, easier binding).
- Negative modulators shift it right.
- Homotropic effector = substrate itself acts as modulator. Heterotropic = a different molecule.
Two models explain cooperativity:
| Model | Concept |
|---|---|
| Concerted (MWC, Monod-Wyman-Changeux) | All subunits flip together between T (tense) and R (relaxed) states; symmetry preserved |
| Sequential (KNF, Koshland) | Subunits change one at a time; "induced fit" propagates |
High-yield: The classic regulated allosteric enzyme of glycolysis is phosphofructokinase-1 (PFK-1) — inhibited by ATP and citrate, activated by AMP and fructose-2,6-bisphosphate (the most potent allosteric activator). Aspartate transcarbamoylase (ATCase) is the textbook allosteric enzyme of pyrimidine synthesis.
Distinguish allosteric regulation from covalent modification (phosphorylation/dephosphorylation, e.g. glycogen phosphorylase) and from feedback inhibition (end product inhibits the committed/first step, usually allosterically).
Factors affecting enzyme activity
- Temperature: rate rises with temperature until the optimum (~37 °C in humans), then falls sharply due to denaturation. Q10 (temperature coefficient) ≈ 2 — rate roughly doubles per 10 °C rise within the working range.
- pH: bell-shaped curve with an optimum (pepsin ~2, salivary amylase ~6.8, trypsin/alkaline phosphatase ~8–10, acid phosphatase ~5).
- Enzyme & substrate concentration as above.
Isoenzymes & enzymes in clinical diagnosis
Isoenzymes have different physical properties (electrophoretic mobility, Km) and tissue distribution, allowing organ-specific diagnosis. This sub-topic alone yields several MCQs every year.
Lactate dehydrogenase (LDH)
LDH is a tetramer of two subunit types, H (heart) and M (muscle), giving 5 isoenzymes:
| Isoenzyme | Composition | Predominant tissue |
|---|---|---|
| LDH-1 | H₄ | Heart, RBC, kidney |
| LDH-2 | H₃M | Reticuloendothelial system (normally highest in serum) |
| LDH-3 | H₂M₂ | Lung, lymphoid |
| LDH-4 | HM₃ | — |
| LDH-5 | M₄ | Liver, skeletal muscle |
High-yield: A "flipped LDH" pattern (LDH-1 > LDH-2) indicates myocardial infarction (and also haemolysis/megaloblastic anaemia). LDH peaks late (3–4 days) after MI; troponin is the modern marker of choice, but the flip remains a classic exam fact. Normally LDH-2 > LDH-1.
Creatine kinase (CK / CPK)
A dimer of M and B subunits — 3 isoenzymes:
| Isoenzyme | Subunits | Source / significance |
|---|---|---|
| CK-MM (CK-3) | MM | Skeletal muscle (also myopathies, exercise) |
| CK-MB (CK-2) | MB | Cardiac muscle — rises 4–6 h post-MI, peaks 24 h, useful for re-infarction |
| CK-BB (CK-1) | BB | Brain, smooth muscle |
High-yield: CK-MB is the classic cardiac isoenzyme; its early rise and fall (normalises in 48–72 h) makes it valuable for detecting re-infarction, where troponin (which stays elevated 7–10 days) is less helpful.
Alkaline phosphatase (ALP)
ALP isoenzymes arise from bone, liver, intestine, placenta, kidney. Distinguish causes of a raised ALP:
| Cause | Clue |
|---|---|
| Bone (osteoblastic activity) | Paget's disease (highest ALP levels), osteomalacia, rickets, healing fracture, bony metastases (osteoblastic) |
| Hepatobiliary | Obstructive/cholestatic jaundice (ALP ↑↑ with ↑ GGT) |
| Physiological | Pregnancy (placental), growing children |
| Regan isoenzyme | Placental-like ALP — a tumour marker (carcinoplacental) |
High-yield: A raised ALP with a raised GGT points to a hepatobiliary source; a raised ALP with normal GGT points to bone. Use GGT (or 5'-nucleotidase) to localise the source.
Other diagnostic enzymes
| Enzyme | Clinical use |
|---|---|
| AST (SGOT) / ALT (SGPT) | Hepatocellular injury; AST:ALT > 2 suggests alcoholic liver disease; ALT > AST in viral hepatitis |
| Amylase & Lipase | Acute pancreatitis; lipase is more specific and stays elevated longer |
| GGT | Cholestasis & alcohol marker; most sensitive for biliary obstruction |
| Acid phosphatase (PAP) | Prostate cancer (largely replaced by PSA) |
| Troponin T/I | Gold-standard cardiac marker (most sensitive & specific for MI) |
| 5′-nucleotidase | Confirms hepatic origin of raised ALP |
| Aldolase | Muscle disease (Duchenne, polymyositis) |
| ADA | Raised in tuberculous effusions (pleural/peritoneal) |
| Chymotrypsin/trypsinogen | Pancreatic function |
Complications & pitfalls (interpretation traps)
- Macroenzymes (enzyme bound to immunoglobulin, e.g. macro-CK, macroamylase) cause spuriously persistent elevation without disease.
- Haemolysed samples falsely raise LDH, AST and potassium.
- Zinc deficiency lowers ALP (ALP is a zinc metalloenzyme).
- Timing matters: ordering the wrong marker at the wrong time (e.g. LDH too early, CK-MB too late) misleads diagnosis.
Key differentials / "spot-the-difference" sets
- Km vs Vmax: affinity constant vs maximal rate; only Vmax depends on [E].
- Competitive vs uncompetitive: both can be confused — competitive raises Km (Vmax same), uncompetitive lowers both.
- Allosteric vs Michaelis-Menten: sigmoidal vs hyperbolic curve.
- Synthetase (ligase, ATP) vs synthase (lyase, no ATP).
- Coenzyme vs prosthetic group: loose/dissociable vs tight/covalent.
- CK-MB vs troponin: re-infarction detection vs maximal sensitivity/specificity.
Recently asked / exam angle
- Image-based Lineweaver-Burk plots: "Lines intersecting on the Y-axis indicate which type of inhibition?" → Competitive. "Parallel lines?" → Uncompetitive.
- "Inhibitor that decreases both Km and Vmax" → Uncompetitive (lithium on inositol monophosphatase).
- "Enzyme with high Km and not product-inhibited" → Glucokinase.
- "Flipped LDH pattern is seen in" → MI / haemolysis / megaloblastic anaemia.
- "Best index of catalytic efficiency" → kcat/Km (specificity constant).
- "Most potent allosteric activator of PFK-1" → Fructose-2,6-bisphosphate.
- "Suicide substrate" examples → allopurinol, 5-FU, DFMO, aspirin (mechanism-based/irreversible).
- "Raised ALP with normal GGT" → bony origin; "highest ALP" → Paget's disease.
- Numerical: given v = Vmax/2, state the relation Km = [S]; or calculate v from Michaelis-Menten when [S] = Km (v = Vmax/2) or [S] = 2Km, etc.
- "Catalytically perfect enzyme" → catalase, fumarase, acetylcholinesterase (kcat/Km near diffusion limit).
Rapid revision
- Enzymes lower Ea; they do not change ΔG, Keq, or equilibrium position.
- Km = [S] at ½ Vmax; low Km = high affinity; Km is independent of [enzyme].
- Vmax = kcat × [E]; depends on enzyme concentration; kcat/Km is the best efficiency index.
- Lineweaver-Burk: Y-intercept = 1/Vmax, X-intercept = −1/Km, slope = Km/Vmax.
- Competitive: Km ↑, Vmax same, lines meet on Y-axis, reversed by excess substrate.
- Non-competitive: Vmax ↓, Km same, lines meet on X-axis.
- Uncompetitive: both Km and Vmax ↓, parallel lines (lithium classic).
- Glucokinase = high Km, not product-inhibited, acts post-prandially.
- Allosteric enzymes give sigmoidal curves; PFK-1 activated by F-2,6-BP & AMP, inhibited by ATP/citrate.
- Flipped LDH (LDH-1 > LDH-2) = MI / haemolysis; LDH-5 = liver/muscle.
- CK-MB = cardiac, best for re-infarction; troponin = most sensitive/specific for MI.
- Raised ALP + GGT = hepatobiliary; raised ALP with normal GGT = bone (Paget's = highest ALP).