Biochemistry chapters
Clinical enzymology
How enzymes work and are measured: classification, coenzymes, Michaelis-Menten kinetics (Km and Vmax) and the Lineweaver-Burk plot, competitive and non-competitive inhibition with drug examples, regulation, isoenzymes, and the enzymes used to diagnose heart, liver, pancreas, muscle and bone disease, with worked calculations.
- Enzymes speed reactions by lowering activation energy without changing equilibrium; Km is the substrate concentration at half Vmax (a low Km = high affinity), and Vmax is the maximal rate when the enzyme is saturated; the Lineweaver-Burk plot (1/v against 1/[S]) has a y-intercept of 1/Vmax and an x-intercept of −1/Km.
- Competitive inhibitors resemble the substrate: Km rises, Vmax unchanged (overcome by more substrate: methotrexate, statins, ethanol for methanol poisoning); non-competitive inhibitors bind elsewhere: Vmax falls, Km unchanged; uncompetitive inhibitors lower both; irreversible inhibitors (aspirin on COX, organophosphates on cholinesterase) destroy activity.
- Diagnostic enzymes: troponin (most specific for myocardial injury), CK-MB (heart; useful for re-infarction), ALT (liver-specific), AST (liver, heart, muscle; AST > ALT in alcoholic liver disease), ALP (bone, biliary obstruction), GGT (biliary, alcohol), lipase and amylase (acute pancreatitis), CK-MM (muscle injury, muscular dystrophy).
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Basics
Enzymes are biological catalysts, mostly proteins (some are RNA: ribozymes). They:
- Lower the activation energy of a reaction.
- Do not change the equilibrium constant or the free energy change (ΔG).
- Are specific and are not used up.
Cofactors: metal ions (Zn²⁺ in carbonic anhydrase, Mg²⁺ with ATP, Fe, Cu) or coenzymes (organic, often from B vitamins: NAD⁺ from niacin, FAD from riboflavin, TPP from thiamine, PLP from B6, CoA from pantothenic acid, biotin, tetrahydrofolate). A tightly bound coenzyme is a prosthetic group. Apoenzyme + cofactor = holoenzyme.
Classification (by reaction type)
| Class | Reaction | Example |
|---|---|---|
| 1. Oxidoreductases | Oxidation-reduction | Dehydrogenases (LDH), oxidases |
| 2. Transferases | Move groups | Aminotransferases (ALT), kinases |
| 3. Hydrolases | Hydrolysis (break bonds with water) | Digestive enzymes, phosphatases, lipase |
| 4. Lyases | Break bonds without water or oxidation | Aldolase, decarboxylases |
| 5. Isomerases | Rearrange | Phosphohexose isomerase |
| 6. Ligases (synthetases) | Join molecules using ATP | DNA ligase, carboxylases |
| 7. Translocases | Move ions or molecules across membranes | ATP synthase (added recently) |
Order of the classes: O-T-H-L-I-L (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases), then translocases (7).
Factors affecting enzyme activity
- Substrate concentration (rate rises then plateaus at Vmax).
- Enzyme concentration (rate proportional).
- Temperature (rises to an optimum, then the protein denatures).
- pH (each enzyme has an optimum: pepsin about 2, most enzymes about 7.4, alkaline phosphatase about 10, trypsin about 8).
- Inhibitors and activators.
Enzyme kinetics
Michaelis-Menten equation
v = Vmax × [S] ÷ (Km + [S])
- Vmax: maximum velocity when all enzyme is saturated with substrate.
- Km (Michaelis constant): the substrate concentration at which v = ½ Vmax. It reflects affinity: low Km = high affinity (the enzyme reaches half speed at low substrate).
Example: hexokinase (low Km for glucose, high affinity, works at low glucose in all tissues) vs glucokinase (high Km, low affinity, works in the liver and pancreas only when glucose is high after meals).
v = Vmax × [S] ÷ (Km + [S])
Vmax = 100 units, Km = 5 mM
[S] = 5 mM (= Km): v = 100 × 5 ÷ (5 + 5) = 500 ÷ 10 = 50 → ½ Vmax (definition of Km)
[S] = 15 mM (3 × Km): v = 100 × 15 ÷ 20 = 75 → 75% of Vmax
[S] = 45 mM (9 × Km): v = 100 × 45 ÷ 50 = 90 → 90% of Vmax
Low substrate is where Km matters most:
[S] = 1 mM: v = 100 × 1 ÷ 6 ≈ 16.7
Lineweaver-Burk (double reciprocal) plot
Plots 1/v against 1/[S], giving a straight line:
1/v = (Km/Vmax) × (1/[S]) + 1/Vmax
- y-intercept = 1/Vmax
- x-intercept = −1/Km
- slope = Km/Vmax
Line crosses the y-axis at 0.02 → 1/Vmax = 0.02 → Vmax = 1 ÷ 0.02 = 50
Line crosses the x-axis at −0.25 → −1/Km = −0.25 → Km = 1 ÷ 0.25 = 4
Add a competitive inhibitor: y-intercept still 0.02 (Vmax still 50)
x-intercept moves to −0.125 → Km = 1 ÷ 0.125 = 8 (Km doubled)
Add a non-competitive inhibitor instead: x-intercept still −0.25 (Km still 4)
y-intercept moves to 0.04 → Vmax = 1 ÷ 0.04 = 25 (Vmax halved)
Enzyme inhibition
| Type | Binds | Km | Vmax | Overcome by more substrate? | Lineweaver-Burk lines | Examples |
|---|---|---|---|---|---|---|
| Competitive | Active site (resembles substrate) | ↑ | Unchanged | Yes | Meet on the y-axis | Methotrexate (dihydrofolate reductase), statins (HMG-CoA reductase), ethanol vs methanol at alcohol dehydrogenase, sulphonamides (PABA), allopurinol (xanthine oxidase), malonate (succinate dehydrogenase), neostigmine |
| Non-competitive | Different site (enzyme or ES complex) | Unchanged | ↓ | No | Meet on the x-axis | Heavy metals (lead), some allosteric inhibitors |
| Uncompetitive | ES complex only | ↓ | ↓ | No | Parallel lines | Lithium on inositol phosphatase (classic example) |
| Irreversible | Covalent bond | (none commonly tested) | ↓ (enzyme destroyed) | No | (none commonly tested) | Aspirin (COX acetylation), organophosphates (acetylcholinesterase), disulfiram, penicillin (transpeptidase), cyanide (cytochrome oxidase) |
Suicide (mechanism-based) inhibitors: the enzyme converts them into an irreversible inhibitor (allopurinol → alloxanthine, clavulanic acid, 5-fluorouracil, vigabatrin).
Competitive: Km up, Vmax same. Non-competitive: Vmax down, Km same. "Competitive changes Km; non-competitive nets lower Vmax."
Regulation of enzymes
- Allosteric regulation: effectors bind a site other than the active site; allosteric enzymes give a sigmoid (S-shaped) curve, not a hyperbola (often multi-subunit, cooperative). Often the rate-limiting enzyme of a pathway, under feedback inhibition by the end-product (PFK-1 inhibited by ATP and citrate; ALA synthase by haem; HMG-CoA reductase by cholesterol).
- Covalent modification: phosphorylation/dephosphorylation by kinases and phosphatases (glycogen phosphorylase, glycogen synthase).
- Induction and repression of enzyme synthesis (slow: hours to days).
- Zymogens (proenzymes): inactive precursors activated by cleavage: trypsinogen → trypsin (by enteropeptidase), pepsinogen → pepsin, clotting factors.
- Compartmentalisation (urea cycle across mitochondria and cytosol).
Isoenzymes
Isoenzymes catalyse the same reaction but differ in structure, kinetics, charge and tissue distribution, so they help identify the damaged organ. Separated by electrophoresis.
| Enzyme | Isoenzymes and sources |
|---|---|
| Creatine kinase (CK) | CK-MM (CK3): skeletal muscle; CK-MB (CK2): heart; CK-BB (CK1): brain |
| Lactate dehydrogenase (LDH) | Five: LDH-1 (H4): heart and red cells; LDH-2 normally highest; LDH-5 (M4): liver and skeletal muscle. In MI, LDH-1 exceeds LDH-2 ("flipped pattern"); now outdated for MI diagnosis |
| Alkaline phosphatase (ALP) | Liver, bone, placenta (heat-stable; Regan isoenzyme in some cancers), intestine |
Diagnostic (clinical) enzymes
| Condition | Enzymes and markers |
|---|---|
| Myocardial infarction | Cardiac troponin I or T: the most sensitive and specific; rises within about 3–4 hours (high-sensitivity assays earlier), peaks around 24 hours, stays raised 7–10 days (troponin T up to about 2 weeks). CK-MB rises in 4–6 hours, normal in 2–3 days (useful for re-infarction). Myoglobin rises earliest but is not specific |
| Liver: hepatocellular injury | ALT (more liver-specific) and AST; very high (thousands) in viral or toxic hepatitis (paracetamol) and ischaemic hepatitis. AST:ALT above 2 suggests alcoholic liver disease |
| Liver: cholestasis | ALP and GGT both ↑ (GGT confirms the ALP is from the liver); GGT also rises with alcohol and enzyme-inducing drugs |
| Bone | ALP ↑ in Paget disease (very high), osteomalacia/rickets, bone metastases (osteoblastic), healing fractures, hyperparathyroidism; normal in osteoporosis and myeloma |
| Acute pancreatitis | Lipase (more specific, stays raised longer) and amylase (rises within hours; more than 3 times the upper limit supports the diagnosis; also raised in mumps, perforated ulcer, renal failure, macroamylasaemia) |
| Muscle damage | CK (CK-MM): rhabdomyolysis, crush injury, Duchenne muscular dystrophy (very high), myositis, statins, IM injections, strenuous exercise |
| Prostate cancer | PSA (now replaces prostatic acid phosphatase) |
| Organophosphate poisoning, suxamethonium apnoea | Plasma (pseudo)cholinesterase ↓ |
| Haemolysis | LDH ↑ |
Expect: what enzymes do and do not change, enzyme classes, coenzymes from vitamins, the definition and meaning of Km and Vmax, Michaelis-Menten and Lineweaver-Burk intercepts, competitive vs non-competitive vs uncompetitive inhibition with drug examples, irreversible and suicide inhibitors, allosteric enzymes (sigmoid curve), zymogens, isoenzymes of CK and LDH, troponin vs CK-MB timing, ALT vs AST, ALP and GGT, lipase vs amylase.
Quick revision
- Enzymes lower activation energy; do not change equilibrium.
- Km = [S] at ½ Vmax; low Km = high affinity (hexokinase low Km, glucokinase high Km).
- Lineweaver-Burk: y-intercept 1/Vmax, x-intercept −1/Km.
- Competitive: Km ↑, Vmax same (methotrexate, statins, ethanol for methanol).
- Non-competitive: Vmax ↓, Km same. Uncompetitive: both ↓.
- Irreversible: aspirin, organophosphates. Allosteric: sigmoid curve.
- Troponin: most specific for MI. CK-MB: re-infarction. ALT: liver-specific. ALP + GGT: cholestasis. Lipase: pancreatitis.
Written by The House Job from standard biochemistry references (Lippincott Illustrated Reviews: Biochemistry).
FAQ
What does a low Km mean?
That the enzyme has a high affinity for its substrate and reaches half its maximum rate at a low substrate concentration.
How does a competitive inhibitor affect Km and Vmax?
It raises Km (lower apparent affinity) but leaves Vmax unchanged, because enough substrate can outcompete it.
Which marker is most specific for myocardial infarction?
Cardiac troponin (I or T).
Which liver enzyme is most specific for liver cell damage?
Alanine aminotransferase (ALT).