Enzymology — Enzyme Classification, Kinetics and Inhibition
🟢 Lite — Quick Review (1h–1d)
Rapid summary for last-minute revision before your exam.
Enzymology is the study of enzymes — biological catalysts that accelerate chemical reactions without being consumed. INI CET frequently tests enzyme kinetics (Michaelis-Menten), inhibition types (competitive vs non-competitive), and co-factors. A single理解了Km和Vmax图表能帮你解决大部分题目。
High-Yield Facts for INI CET:
- Enzyme classification: 6 major classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases) — remember with mnemonic “OHL TIL”
- Km = substrate concentration at Vmax/2; lower Km = higher affinity
- Competitive inhibition: Km increases, Vmax unchanged (same maximum velocity if substrate is infinite)
- Non-competitive inhibition: Vmax decreases, Km unchanged
- Co-factors: Zn²⁺ (carbonic anhydrase), Fe²⁺ (cytochrome oxidase), Mg²⁺ (ATP-dependent enzymes), biotin (carboxylation reactions)
⚡ Exam tip: In a Lineweaver-Burk plot, competitive inhibition shifts the Lineweaver-Burk line upward on the y-axis (1/Vmax unchanged, -1/Km more negative). Non-competitive shifts horizontally (1/Vmax more negative, -1/Km unchanged).
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Enzymology — INI CET (AIIMS PG) Study Guide
Enzyme Structure and Classification
Enzyme Structure:
- Apoenzyme: Protein part of the enzyme (core catalyst)
- Cofactor: Non-protein component required for activity
- Holoenzyme: Active enzyme = apoenzyme + cofactor
- Prosthetic group: Tightly bound cofactor (e.g., heme in catalase)
- Coenzyme: Loosely bound, diffusible carrier of chemical groups (e.g., NAD⁺, FAD, coenzyme A)
The 6 Enzyme Classes (with mnemonics):
| Class | Reaction Type | Example |
|---|---|---|
| Oxidoreductases | Redox reactions (electron transfer) | LDH (lactate ↔ pyruvate), cytochrome oxidase |
| Transferases | Transfer of groups (CH₃, NH₂, PO₄, etc.) | ALT, AST (transamination), hexokinase (phosphate transfer) |
| Hydrolases | Cleavage by water (hydrolysis) | Pepsin, trypsin, lipase, alkaline phosphatase |
| Lyases | Non-hydrolytic cleavage (adds/removes double bonds) | Aldolase (splits fructose-1,6-bisphosphate), enolase |
| Isomerases | Rearrangement of atoms within a molecule | Triose phosphate isomerase, phosphoglucomutase |
| Ligases | Joining of two molecules (requires ATP) | DNA ligase, synthetases, carboxylases |
Mnemonic: “OxeD TLy IL” = “Oh Dear, Tell LY In Limerick”
Enzyme Kinetics — Michaelis-Menten
The Michaelis-Menten Equation: v = Vmax × [S] / (Km + [S])
Where:
- v = initial reaction velocity
- Vmax = maximum velocity (when all enzyme is saturated)
- [S] = substrate concentration
- Km = Michaelis constant (substrate concentration at v = Vmax/2)
Significance of Km:
- Km is the substrate concentration at which velocity is half of Vmax
- Lower Km = higher enzyme-substrate affinity (fewer substrate molecules needed)
- Km is independent of enzyme concentration
- Km equals [S] at which v = Vmax/2
Lineweaver-Burk (Double Reciprocal) Plot:
- 1/v vs 1/[S] gives a straight line
- Y-intercept = 1/Vmax
- X-intercept = -1/Km
- Slope = Km/Vmax
Enzyme Units:
- 1 International Unit (IU) = amount catalyzing conversion of 1 μmol substrate/minute at 25°C
- Katal (SI unit) = 1 mol/second (1 IU = 16.67 nkat)
🔴 Extended — Deep Study (3mo+)
Comprehensive coverage for students on a longer study timeline.
Enzyme Inhibition
1. Competitive Inhibition:
- Inhibitor resembles substrate and competes for active site
- Effect: Km increases (apparent decrease in affinity), Vmax unchanged
- Can be overcome by increasing substrate concentration
- Example: Methotrexate (structurally similar to folic acid) inhibits dihydrofolate reductase
- Lineweaver-Burk: Y-intercept same, X-intercept more negative (more negative -1/Km)
2. Non-Competitive Inhibition:
- Inhibitor binds to a site other than the active site (allosteric site)
- Cannot be overcome by increasing substrate
- Effect: Vmax decreases, Km unchanged (all enzyme can still bind substrate with same affinity)
- Example: Heavy metals (Hg²⁺, Pb²⁺) binding to enzyme -SH groups; cyanide inhibits cytochrome oxidase
- Lineweaver-Burk: Y-intercept increases (1/Vmax increases), X-intercept unchanged
3. Uncompetitive Inhibition:
- Inhibitor binds only to enzyme-substrate complex
- Effect: Both Km and Vmax decrease
- Lineweaver-Burk: Lines intersect to the LEFT of Y-axis (decreased Vmax, decreased Km)
4. Mixed Inhibition:
- Inhibitor can bind both free enzyme and enzyme-substrate complex
- Effect: Vmax decreases, Km may increase or decrease
5. Irreversible Inhibition:
- Covalent binding to enzyme (or tight non-covalent)
- Examples: Aspirin (acetylates cyclooxygenase), organophosphates (inhibit acetylcholinesterase), nerve agents
Allosteric Regulation:
- Modulator binds at site other than active site → changes enzyme conformation → affects activity
- Homotropic: Same molecule is both substrate and modulator (e.g., O₂ binding to hemoglobin)
- Heterotropic: Different molecule modulates activity (e.g., ATP/ADP modulating phosphofructokinase)
- Allosteric enzymes show sigmoid (S-shaped) kinetics rather than hyperbolic Michaelis-Menten
- Fitted by Hill equation; Hill coefficient (n) > 1 indicates cooperativity
Enzyme Catalysis Mechanisms
Catalytic Strategies Used by Enzymes:
- Proximity and orientation effects: Bring substrates together in correct orientation
- Acid-base catalysis: Donation/acceptance of protons (e.g., chymotrypsin uses His as a base)
- Covalent catalysis: Formation of enzyme-substrate intermediate (e.g., serine proteases)
- Metal ion catalysis: Participate in redox reactions or stabilize charges
- Electrostatic catalysis: Stabilize transition states
Serine Proteases (Chymotrypsin, Trypsin, Elastase):
- Use the “catalytic triad”: Ser-His-Asp
- Ser acts as nucleophile, forming acyl-enzyme intermediate
- His acts as general base/acid
- Asp stabilizes His through hydrogen bonding
- Mechanism: Substrate binds → nucleophilic attack by Ser → acyl-enzyme intermediate → hydrolysis by water → release of products
Cofactors and Their Functions:
| Cofactor | Function | Enzyme |
|---|---|---|
| NAD⁺/NADH | Electron (hydride) carrier | LDH, malate dehydrogenase |
| FAD/FADH₂ | Electron carrier | Succinate dehydrogenase |
| NADP⁺/NADPH | Reducing power (biosynthesis) | G6PD, glutathione reductase |
| Coenzyme A | Acyl group carrier | Fatty acid synthesis, TCA cycle |
| Biotin | Carboxylation (CO₂ transfer) | Pyruvate carboxylase, acetyl-CoA carboxylase |
| Thiamine pyrophosphate (TPP) | Aldehyde transfer | Pyruvate dehydrogenase, transketolase |
| Pyridoxal phosphate (PLP) | Transamination, decarboxylation | ALT, AST, decarboxylases |
| Cobalamin (B12) | Methyl group transfer | Methionine synthase |
| Tetrahydrofolate | One-carbon transfers | DNA synthesis, purine synthesis |
| Zn²⁺ | Structural and catalytic | Carbonic anhydrase, alcohol dehydrogenase |
| Mg²⁺ | ATP binding and transfer | Kinases, phosphatases |
| Mn²⁺ | Oxidoreductases | Arginase, pyruvate carboxylase |
| Se | Redox (oxidoreductases) | Glutathione peroxidase |
Clinical Enzymology
Serum Enzyme Markers:
| Enzyme | Elevated In | Clinical Significance |
|---|---|---|
| ALT (SGPT) | Liver disease (hepatitis, cirrhosis) | Hepatocellular damage |
| AST (SGOT) | Liver disease, MI, muscle injury | Hepatocellular and cardiac damage |
| CK (CPK) | Muscle damage (MI, rhabdomyolysis, DMD) | Cardiac/muscle injury |
| LDH | MI, hemolysis, liver disease, malignancy | Non-specific tissue damage |
| Alkaline phosphatase | Bone disease, cholestasis, pregnancy | Hepatobiliary/bone pathology |
| GGT | Cholestasis, alcoholism | Hepatobiliary disease |
| Amylase | Pancreatitis, salivary gland disease | Acute pancreatitis |
| Lipase | Pancreatitis | More specific for pancreatitis than amylase |
| Troponin I/T | Myocardial infarction | Gold standard for MI |
| CPK-MB | MI | Cardiac-specific isoenzyme |
Isoenzymes:
- CK-MM: Skeletal muscle (85-90% of total in normal serum)
- CK-MB: Cardiac muscle (elevated in MI)
- CK-BB: Brain (rarely elevated in serum; used in research)
- LDH-1 (H4): Heart, RBCs (elevated in MI)
- LDH-5 (M4): Liver, skeletal muscle (elevated in liver damage)
- Amylase: Pancreatic (P-type) vs salivary (S-type) — can be separated by electrophoresis
Enzyme Therapy:
- Streptokinase/streptokinase derivatives: Clot-busting (plasminogen activator)
- Asparaginase: Treats ALL (deprives leukemic cells of asparagine)
- PEG-asparaginase: Longer-acting form
⚡ INI CET High-Yield: Remember that ALT > AST suggests viral hepatitis; AST > ALT suggests alcoholic liver disease, cirrhosis, or MI. Elevated GGT with elevated ALP suggests cholestasis rather than bone disease.
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