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ENZYMES - 1st Year Biochemistry Exam Notes

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HOW TO USE THESE NOTES

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LONG ANSWER TOPICS

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1. Enzyme Classification (IUB Classification)

Naming System

• Recommended (trivial) name - ends in "-ase" attached to substrate (e.g., glucosidase, urease) or describes the action (e.g., lactate dehydrogenase). Some retain old trivial names - trypsin, pepsin.
• Systematic name - attached to a complete description of the reaction, including all substrates (e.g., lactate:NAD+ oxidoreductase = EC 1.1.1.27).

Six IUB Classes

Mnemonic: "Old Tigers Hate Losing In Life" = Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases

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2. Mechanism of Enzyme Action

How Enzymes Lower Activation Energy

• Enzymes provide an alternate reaction pathway with a lower activation energy (Ea).
• They do NOT change the equilibrium of the reaction - only the rate at which equilibrium is achieved.
• Reactions proceed 10³ to 10⁸ times faster than uncatalyzed reactions.

Active Site

• A special pocket or cleft formed by folding of the protein.
• Contains amino acid residues that participate in substrate binding and catalysis.
• The enzyme is much larger than the substrate; only a few amino acids form the active site.

Steps in Catalysis:

1. Substrate (S) binds to the active site → Enzyme-Substrate (ES) complex is formed.
2. Conformational change in enzyme occurs (Induced Fit Model).
3. ES complex is converted to Enzyme-Product (EP) complex.
4. Product is released; free enzyme is regenerated.

Two Models of Enzyme-Substrate Interaction:

Mechanisms Used in Catalysis:

1. Acid-base catalysis - amino acid side chains (e.g., histidine) donate/accept protons.
2. Covalent catalysis - temporary covalent bond formed between enzyme and substrate.
3. Metal ion catalysis - metal ions (Zn²+, Mg²+) stabilize negative charges or act as Lewis acids.
4. Proximity and orientation effects - enzyme holds substrates in correct orientation.

Michaelis-Menten Kinetics:

• Plot of initial reaction velocity (v₀) vs. substrate concentration [S] gives a hyperbolic curve.
• Vmax = maximum velocity when all enzyme molecules are saturated with substrate.
• Km (Michaelis constant) = [S] at which v₀ = ½ Vmax. It reflects enzyme's affinity for substrate (low Km = high affinity).
• Lineweaver-Burk (double-reciprocal) plot = 1/v₀ vs. 1/[S] - a straight line used to calculate Km and Vmax.
• Turnover number (kcat) = number of substrate molecules converted to product per enzyme per second = 10² to 10⁴ /sec.

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3. Enzyme Inhibition

A. Irreversible Inhibition

• Inhibitor binds through covalent bonds - cannot be displaced.
• Example: Lead inhibits ferrochelatase (heme synthesis) by binding -SH (cysteine) groups.
• Example: Organophosphates (nerve agents, insecticides) irreversibly inhibit acetylcholinesterase.
• Example: Aspirin irreversibly inhibits cyclooxygenase (COX), blocking prostaglandin synthesis.

B. Reversible Inhibition

1. Competitive Inhibition

• Inhibitor has structural similarity to substrate.
• Competes for the same active site.
• Km increases (apparent); Vmax is unchanged (can be overcome by excess substrate).
• On Lineweaver-Burk plot: lines intersect on the y-axis (same Vmax, different x-intercepts).
• Example: Methotrexate competes with folate at dihydrofolate reductase. Statins compete with HMG-CoA at HMG-CoA reductase.

2. Noncompetitive Inhibition

• Inhibitor binds to a site different from the active site (allosteric site) on both free enzyme and ES complex.
• Does NOT affect substrate binding, so Km is unchanged.
• Vmax decreases - cannot be reversed by adding more substrate.
• On Lineweaver-Burk plot: lines intersect on the x-axis (same Km, different y-intercepts).

3. Uncompetitive Inhibition

• Inhibitor binds only to the ES complex (not free enzyme).
• Both Km and Vmax decrease proportionally.
• On Lineweaver-Burk plot: lines are parallel (same slope).

Summary Table

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4. Regulation of Enzyme Activity

A. Allosteric Regulation

• Allosteric enzymes are composed of multiple subunits and do NOT follow Michaelis-Menten kinetics - they show a sigmoidal curve.
• Regulated by effectors that bind at the allosteric site (not the active site).
• Positive effectors - increase enzyme activity (activate).
• Negative effectors - decrease enzyme activity (inhibit).
• Example: Phosphofructokinase-1 (PFK-1) is positively regulated by AMP/ADP and negatively regulated by ATP/citrate.

B. Covalent Modification (Phosphorylation/Dephosphorylation)

• Enzymes can be activated or inactivated by adding or removing phosphate groups (via kinases and phosphatases).
• Example: Glycogen phosphorylase is activated by phosphorylation (by protein kinase A) and inactivated by dephosphorylation.
• Example: Glycogen synthase is inactivated by phosphorylation.

C. Zymogen (Proenzyme) Activation

• Some enzymes are synthesized as inactive precursors (zymogens/proenzymes).
• Activated by proteolytic cleavage of a peptide bond.
• Examples: Pepsinogen → Pepsin; Trypsinogen → Trypsin; Chymotrypsinogen → Chymotrypsin; Prothrombin → Thrombin.

D. Enzyme Induction/Repression

• Enzyme induction - substrate or hormone induces increased synthesis of enzyme (gene expression).
• Enzyme repression - end-product inhibits enzyme synthesis.
• Example: HMG-CoA reductase is induced by decreased intracellular cholesterol.

E. Feedback (End-Product) Inhibition

• The end product of a metabolic pathway inhibits an early enzyme in the pathway (usually the committed step).
• This is a form of negative allosteric regulation.
• Example: ATP inhibits phosphofructokinase in glycolysis.

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SHORT ANSWER TOPICS

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5. Coenzymes

• Apoenzyme = protein portion alone (inactive).
• Coenzyme/Cofactor = nonprotein component.
• Holoenzyme = apoenzyme + coenzyme = active enzyme.
• Prosthetic group = cofactor covalently or very tightly bound to the enzyme (e.g., heme in cytochromes, biotin).

Two Classes:

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6. Factors Affecting Enzyme Activity

1. Substrate Concentration [S]

• As [S] increases, v₀ increases until Vmax is reached (enzyme saturation).
• Follows Michaelis-Menten kinetics (hyperbolic curve for most enzymes).
• Allosteric enzymes show sigmoidal curve.

2. Temperature

• Velocity increases with temperature (more substrate molecules have enough energy).
• Optimum temperature for most human enzymes = 35-40°C (optimal at 37°C).
• Temperatures above 40°C cause enzyme denaturation → velocity decreases.
• Thermophilic bacteria have optimal temp of 70°C.

3. pH

• Each enzyme has an optimal pH at which activity is maximal.
• Changes in pH:
  • Alter ionization state of active site amino acids.
  • Extremes denature the enzyme.
• Examples: Pepsin - optimal pH 2; Trypsin - optimal pH 8; Alkaline phosphatase - optimal pH 9-10.

4. Enzyme Concentration

• Velocity increases proportionally with enzyme concentration (as long as substrate is in excess).

5. Inhibitors and Activators (as covered above)

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7. Enzymes in Liver Diseases

• ALT is more specific for liver damage than AST because it is found predominantly in the liver.
• In viral hepatitis - ALT > AST.
• In alcoholic hepatitis - AST > ALT (ratio >2:1).
• In obstructive jaundice - ALP, GGT, and 5'NT are raised.

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8. Cardiac Markers (Enzymes in Cardiac Disease)

Creatine Kinase (CK) and its Isoforms

• CK is a dimer of two subunits: M (muscle) and B (brain).
• Three isoenzymes:
  • CK-BB (CK-1) - Brain
  • CK-MB (CK-2) - Heart (most significant for MI)
  • CK-MM (CK-3) - Skeletal muscle

• CK-MB rises 4-8 hours after MI, peaks at 18-24 hours, returns to normal by 48-72 hours.

Lactate Dehydrogenase (LDH) Isoenzymes

• LDH has 5 isoenzymes (LDH₁ to LDH₅).
• LDH₁ > LDH₂ is characteristic of MI (normally LDH₂ > LDH₁ - this reversal is called "LDH flip").
• LDH rises later and stays elevated longer than CK.

Troponins (Gold Standard)

• Cardiac-specific isoforms of troponin T (cTnT) and troponin I (cTnI) are the current gold standard for MI diagnosis.
• Appear in plasma 4-6 hours after MI.
• Peak at 24-36 hours.
• Remain elevated for 3-10 days.
• More sensitive and specific than CK-MB.

AST (SGOT)

• Was historically used but less specific.
• Rises 8-12 hours after MI, peaks at 24-36 hours.

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9. Isoenzymes

• Same reaction, but different Km, Vmax, and electrophoretic mobility.
• Separated by electrophoresis (they have different charges).
• Different tissue distribution - makes them useful as organ-specific diagnostic markers.

Important Examples:

• Tetramer made of two types of subunits: H (heart) and M (muscle).
• 5 isoenzymes: LDH₁ (H₄) to LDH₅ (M₄).

• MI: LDH₁ > LDH₂ ("LDH flip").
• Liver disease: LDH₄ and LDH₅ elevated.

• CK-MB elevated in MI (as above).

• Liver isoform vs. bone isoform - helps differentiate liver vs. bone disease.

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10. Enzyme Specificity

Types of Specificity:

1. Absolute specificity - enzyme acts on only one substrate and catalyzes only one reaction.
   • Example: Urease acts only on urea; glucose oxidase acts only on D-glucose.
2. Group specificity - enzyme acts on molecules with a particular chemical group (functional group), regardless of the rest of the molecule.
   • Example: Hexokinase acts on hexoses (D-glucose, D-fructose, D-mannose).
3. Linkage specificity - enzyme acts on a specific type of chemical bond, regardless of the structure of the rest of the molecule.
   • Example: Lipases cleave ester bonds; peptidases cleave peptide bonds.
4. Stereospecificity - enzyme acts on only one stereoisomer (D or L form, or cis/trans isomers).
   • Example: L-amino acid oxidase acts only on L-amino acids, not D-amino acids.
   • This is the most important type of specificity in biological systems.

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11. Uses of Enzymes

A. Diagnostic Uses (Most Important)

• Serum enzyme levels reflect tissue damage (see liver diseases, cardiac markers).
• Key diagnostic enzymes:

B. Therapeutic Uses

• Streptokinase, tPA (tissue plasminogen activator) - dissolve blood clots in MI and stroke.
• Hyaluronidase - helps drug absorption by breaking down hyaluronic acid.
• Asparaginase - used in leukemia (cancer cells depend on asparagine).
• Trypsin, chymotrypsin - debridement of wounds.
• Pancreatin (lipase, amylase, protease) - enzyme replacement in pancreatic insufficiency.
• Urokinase - thrombolytic therapy.

C. Industrial Uses

• Amylase - bread making, brewing.
• Proteases - meat tenderizers, detergents.
• Lactase - lactose-free milk production.
• DNA ligase, restriction enzymes - recombinant DNA technology.

D. Research Uses

• ELISA (Enzyme-Linked Immunosorbent Assay) - uses enzyme-labeled antibodies to detect antigens/antibodies.
• PCR - uses thermostable DNA polymerase (Taq polymerase).

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QUICK REVISION TABLE

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Source: Lippincott's Illustrated Reviews: Biochemistry, 8th Edition; Basic Medical Biochemistry: A Clinical Approach, 6th Edition.