Enzymes and enzyme kinetics
Enzymes: kinetics, inhibition, regulation, key enzymes and clinical enzymology
Enzymes for FCPS Part 1: coenzymes, Km and Vmax, Lineweaver–Burk plots, inhibition types, regulation and key rate-limiting enzymes. Ends with clinical enzymology for heart, liver, pancreas, bone and muscle.
One of the most tested topics in past papers
How enzymes work
- Enzymes lower activation energy, speeding the approach to equilibrium. They do not change free energy change, the equilibrium constant or the direction of a reaction, and are not consumed.
- Apoenzyme (protein) plus cofactor gives the active holoenzyme.
- Metal cofactors: zinc (carbonic anhydrase, alcohol dehydrogenase), magnesium (with ATP), copper (cytochrome oxidase, lysyl oxidase), selenium (glutathione peroxidase), molybdenum (xanthine oxidase), iron (catalase, cytochromes).
| Coenzyme | Vitamin source | Example enzyme |
|---|---|---|
| NAD, NADP | Niacin (B3) | Dehydrogenases |
| FAD, FMN | Riboflavin (B2) | Succinate dehydrogenase |
| Thiamine pyrophosphate | Thiamine (B1) | Pyruvate dehydrogenase, transketolase |
| Pyridoxal phosphate | Pyridoxine (B6) | Aminotransferases, decarboxylases |
| Coenzyme A | Pantothenic acid (B5) | Acyl group carrier |
| Biotin | Biotin (B7) | Carboxylases |
| Tetrahydrofolate | Folate | One-carbon transfers |
| Methylcobalamin | B12 | Methionine synthase |
- Classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases.
Michaelis–Menten kinetics
- Vmax is the rate when all enzyme molecules are saturated with substrate; it is proportional to the amount of enzyme. Saturation is the same idea as the transport maximum (Tm) of carrier-mediated transport in the kidney.
- Km is the substrate concentration at half Vmax; a low Km means high affinity.
- Lineweaver–Burk (double reciprocal) plot: y-intercept is 1/Vmax, x-intercept is -1/Km, slope is Km/Vmax.
- Allosteric enzymes with several subunits (phosphofructokinase-1, aspartate transcarbamoylase) give a sigmoid substrate–velocity curve because binding at one site changes affinity at others (cooperativity), like haemoglobin's oxygen curve.
- Each enzyme has an optimum temperature (about 37 °C) and pH (pepsin about 2, trypsin about 8, alkaline phosphatase about 10).
Inhibition
| Type | Binds | Km | Vmax | Overcome by more substrate? | Examples |
|---|---|---|---|---|---|
| Competitive | Active site (free enzyme) | Rises | Unchanged | Yes | Methotrexate (DHFR), statins, ethanol or fomepizole against methanol on alcohol dehydrogenase, sulphonamides (PABA), malonate (succinate dehydrogenase) |
| Non-competitive | Separate site on enzyme or complex | Unchanged | Falls | No | Heavy metals |
| Uncompetitive | Enzyme–substrate complex only | Falls | Falls | No | Lithium on inositol monophosphatase |
| Irreversible | Covalent bond | – | Falls | No | Aspirin (acetylates COX), organophosphates (acetylcholinesterase), omeprazole (H+/K+ ATPase), penicillin (transpeptidase), disulfiram |
- On Lineweaver–Burk plots, competitive lines meet on the y-axis, non-competitive lines meet on the x-axis, and uncompetitive lines are parallel.
- Suicide inhibitors are turned by the enzyme into an irreversible inhibitor: allopurinol to oxypurinol, clavulanic acid, vigabatrin.
- Oligomycin blocks the proton channel (F0) of ATP synthase, so oxygen use falls; uncouplers do the opposite.
Regulation of enzyme activity
| Mechanism | Speed | Example |
|---|---|---|
| Allosteric (feedback) | Seconds | ATP and citrate inhibit PFK-1; end products inhibit first committed steps |
| Covalent modification | Seconds to minutes | Glucagon raises cAMP, activating protein kinase A, which phosphorylates and activates glycogen phosphorylase (and inactivates glycogen synthase) |
| Induction and repression | Hours to days | Insulin induces glucokinase; drugs induce cytochrome P450 |
| Zymogen activation | Irreversible | Enteropeptidase activates trypsinogen; clotting cascade |
| Compartmentalisation | – | Fatty acid synthesis in cytoplasm, oxidation in mitochondria |
Key enzymes to know
| Pathway | Rate-limiting or key enzyme |
|---|---|
| Glycolysis | Phosphofructokinase-1 |
| Gluconeogenesis | Fructose-1,6-bisphosphatase |
| Glycogen synthesis | Glycogen synthase; branching enzyme forms the alpha-1,6 branch points |
| Glycogen breakdown | Glycogen phosphorylase; debranching enzyme |
| Purine synthesis | Glutamine-PRPP amidotransferase |
| Pyrimidine synthesis | Carbamoyl phosphate synthetase II |
| Cholesterol synthesis | HMG-CoA reductase |
| Reverse cholesterol transport | LCAT esterifies cholesterol in HDL |
| Antioxidant defence | Superoxide dismutase (superoxide to H2O2), catalase and glutathione peroxidase (H2O2 to water) |
- Pyruvate kinase deficiency is the commonest glycolytic enzyme defect causing chronic haemolytic anaemia (G6PD deficiency, in the pentose pathway, is commoner overall). Red cells lack ATP, membranes fail, and 2,3-BPG rises.
- Oxidase enzymes that generate H2O2, and catalase that destroys it, are found in peroxisomes.
- Salivary lactoferrin binds iron, denying it to bacteria; lysozyme attacks their walls.
- Brush border lactase deficiency (common after gastroenteritis that flattens villi) causes osmotic diarrhoea.
- In hypotension the juxtaglomerular cells secrete the enzyme renin, starting the angiotensin cascade.
Second messengers
- Hormones using phospholipase C (Gq) split PIP2 into IP3 (releases calcium) and DAG (activates protein kinase C): alpha-1 adrenergic, ADH on V1, oxytocin, angiotensin II, TRH, GnRH, gastrin, histamine H1, muscarinic M1 and M3.
Glycaemic index
- The glycaemic index compares the rise in blood glucose over two hours after a food containing a fixed amount of carbohydrate with the rise after the same amount of pure glucose (scored 100).
Clinical enzymology
| Marker | Use |
|---|---|
| Myoglobin | Earliest to rise after MI (1–2 hours) but not specific |
| Troponin I or T | Most specific for myocardial injury; rises 3–6 h, peaks 12–24 h, stays raised 7–10 days |
| CK-MB | Rises 4–6 h, normal by 2–3 days: detects reinfarction |
| CK-MM | Skeletal muscle damage (rhabdomyolysis, dystrophy, statins) |
| LDH | Haemolysis, MI (LDH1 above LDH2), lymphoma, megaloblastic anaemia |
| ALT | More liver-specific; very high in acute viral or drug hepatitis |
| AST | Liver, heart, muscle; AST:ALT above 2 suggests alcohol |
| ALP | Cholestasis (with GGT; ALP far above ALT means obstruction), bone growth and disease, placenta |
| GGT | Cholestasis, alcohol, enzyme inducers; tells hepatic from bone ALP |
| Amylase | Acute pancreatitis; rises early and falls within 3–5 days |
| Lipase | More specific for pancreatitis and stays raised longer |
| Pseudocholinesterase | Low in liver disease and organophosphate poisoning; inherited deficiency prolongs suxamethonium |
| Caeruloplasmin | Low in Wilson disease (with Kayser–Fleischer rings; confirm by 24-hour urine copper or liver copper) |
- Isoenzymes catalyse the same reaction with different structure and tissue distribution: LDH1 in heart and red cells, LDH5 in liver and muscle; CK-MM, MB, BB; ALP liver, bone, placental (heat stable) and intestinal.
Remember Competitive raises Km; non-competitive lowers Vmax; uncompetitive lowers both; only competitive is overcome by substrate.
Exam trap An inhibitor that binds only the enzyme–substrate complex lowers both Km and Vmax.
Exam trap A normal amylase on day 5 does not exclude pancreatitis: lipase stays raised longer.
How FCPS asks about this
- Vitamin in coenzyme A: pantothenic acid.
- Allosteric enzyme kinetics: sigmoid (cooperative) curve.
- Glucagon activating glycogen phosphorylase: phosphorylation via cAMP and protein kinase A.
- Enzyme converting superoxide to hydrogen peroxide: superoxide dismutase.
- Commonest glycolytic enzyme defect causing chronic haemolysis: pyruvate kinase deficiency.
- Rate-limiting enzyme of purine synthesis: glutamine-PRPP amidotransferase; enzyme forming glycogen branches: branching enzyme.
- Enzyme esterifying cholesterol in HDL: LCAT.
Test yourself on this topic
5 of the 34 questions in our bank on this topic. Choose an answer to see why every option is right or wrong.
A patient with epigastric pain radiating to the back has a normal serum amylase on day 5 of illness. Which enzyme remains raised longer and is more specific for acute pancreatitis?
Answer: Serum lipase
Amylase rises within hours of acute pancreatitis and falls within 3–5 days; it is also raised in salivary disease, bowel perforation or ischaemia, ruptured ectopic pregnancy, renal failure and macroamylasaemia, so it is less specific. Lipase rises at a similar time but stays elevated longer (up to 1–2 weeks) and is more specific to the pancreas, making it preferred for diagnosis, especially in late presentation. Enzyme levels do not correlate with severity. Clinical enzymology examples: ALT (liver), ALP (bone and biliary), GGT (biliary, alcohol), CK (muscle), troponin (myocardium, a structural protein rather than an enzyme), acid phosphatase (prostate, historically).
Pancreatic enzymes
- Amylase: rises early, falls in 3–5 days, less specific
- Lipase: stays up 1–2 weeks, more specific
- Neither reflects severity
- Amylase also ↑: perforation, ectopic, parotitis, renal failure
Remember Lipase lingers and is loyal to the pancreas.
Why the other options are wrong
- Alkaline phosphatase Bone and biliary disease.
- Creatine kinase Muscle injury.
- Alanine aminotransferase Hepatocellular injury.
- Lactate dehydrogenase only Non-specific.
Which LDH isoenzyme pattern suggests myocardial infarction?
Answer: LDH1 above LDH2 (flipped)
LDH1 (H4) dominates in heart and red cells; LDH5 (M4) in liver and skeletal muscle. Normally LDH2 > LDH1; in MI (and haemolysis) the 'flipped' LDH1 > LDH2 pattern appears 12–24 hours later and persists for days. Troponins have replaced LDH for MI diagnosis.
LDH isoenzymes
- LDH1: heart, red cells
- LDH5: liver, skeletal muscle
- MI: LDH1 > LDH2 (flip)
Remember Heart is number 1; liver is last (5).
Why the other options are wrong
- LDH5 above LDH4 Liver or skeletal muscle injury.
- LDH3 highest Lungs.
- LDH2 above LDH1 (normal) Normal pattern.
- No LDH change LDH rises in MI.
Selenium is an essential component of which antioxidant enzyme?
Answer: Glutathione peroxidase
Glutathione peroxidase (selenium) reduces hydrogen peroxide and lipid peroxides using reduced glutathione. Selenium also forms deiodinases (thyroid hormone activation). Deficiency: Keshan disease (cardiomyopathy) and Kashin–Beck disease. Superoxide dismutase uses Cu/Zn (cytosol) or Mn (mitochondria); catalase uses iron (haem).
Antioxidant enzymes
- Glutathione peroxidase: selenium
- Superoxide dismutase: Cu/Zn or Mn
- Catalase: iron; selenium deficiency → Keshan cardiomyopathy
Remember Selenium guards glutathione.
Why the other options are wrong
- Superoxide dismutase (copper–zinc) Copper and zinc.
- Catalase (iron) Iron.
- Carbonic anhydrase (zinc) Zinc.
- Cytochrome oxidase (copper) Copper.
An infertile man has globozoospermia: his sperm have round heads with no acrosome. They pass through the cumulus poorly and cannot cross the zona pellucida. Which acrosomal enzyme normally digests a path through the zona?
Answer: Acrosin
The acrosome is a cap-like modified lysosome formed from the Golgi apparatus of the spermatid. Hyaluronidase on the sperm surface helps it pass through the hyaluronic acid matrix of the cumulus. After binding to the zona pellucida, the acrosome reaction releases acrosin, a trypsin-like serine protease, which digests a narrow path through the zona for the sperm to reach the oocyte membrane. Sperm without an acrosome need ICSI to fertilise an oocyte.
Acrosome
- Formed from the Golgi apparatus
- Hyaluronidase: through the cumulus
- Acrosin: through the zona
- No acrosome: needs ICSI
Remember Hyaluronidase for the cumulus, acrosin for the zona.
Exam trap Hyaluronidase acts on the cumulus layer; the zona itself is crossed with acrosin.
Why the other options are wrong
- Hyaluronidase Disperses the cumulus cells, not the zona.
- Prostate-specific antigen Prostatic enzyme that liquefies semen.
- Neuraminidase Not the zona-digesting enzyme.
- Collagenase Not an acrosomal enzyme for the zona.
After an overnight fast, a woman at 32 weeks has raised plasma free fatty acids and β-hydroxybutyrate, far more than a non-pregnant woman after the same fast. Which adipose tissue enzyme is most active, released from insulin restraint?
Answer: Hormone-sensitive lipase
Hormone-sensitive lipase hydrolyses stored triglyceride in adipocytes. It is activated by phosphorylation via cAMP and protein kinase A (driven by catecholamines and glucagon), and insulin inhibits it by activating phosphodiesterase and lowering cAMP. In late pregnancy, insulin resistance, falling fasting insulin and hPL all favour lipolysis, so fatty acids flood out and the liver converts them to ketones. This spares glucose for the fetus but makes ketosis come quickly.
Hormone-sensitive lipase
- Breaks down stored triglyceride
- Activated by cAMP/PKA
- Inhibited by insulin
- Pregnancy fasting: FFA and ketones up
Remember Insulin locks the fat store; fasting unlocks it.
Exam trap Lipoprotein lipase and hormone-sensitive lipase respond to insulin in opposite directions.
Why the other options are wrong
- Lipoprotein lipase LPL clears circulating triglyceride into tissues; insulin activates it in fat.
- Acetyl-CoA carboxylase A lipogenic enzyme, activated by insulin.
- Glycerol kinase Adipocytes have little glycerol kinase.
- Fatty acid synthase A lipogenic enzyme, active in the fed state.
Where this sits in the CPSP syllabus
- Paper 1: Biomolecules and metabolism, Enzymes, kinetics, inhibition and clinical enzymology
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