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ENZYMES - Detailed Answers (As per Satyanarayana's Biochemistry)

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Q2. Mechanism of Action of Enzymes [5 Marks]

1. Activation Energy and Transition State

2. Formation of Enzyme-Substrate (ES) Complex

E + S → ES → EP → E + P

3. Chemical Mechanisms Used by Enzymes

• Acid-Base Catalysis: Amino acids (e.g., His, Asp, Glu, Lys) donate or accept protons to stabilize the transition state.
• Covalent Catalysis: A transient covalent bond forms between the enzyme and substrate (e.g., serine proteases - chymotrypsin forms an acyl-enzyme intermediate).
• Metal Ion Catalysis: Metal ions (Zn²⁺, Mg²⁺, Fe²⁺) stabilize negative charges, act as Lewis acids, or participate in oxidation-reduction.
• Proximity and Orientation Effects: The active site brings substrates together in the correct orientation for the reaction to occur, effectively increasing local concentration.
• Strain/Distortion: Binding may induce strain in the substrate, making it more reactive by destabilizing bonds.

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Q3. Factors Affecting Enzyme Activity [10 Marks]

1. Substrate Concentration [S]

• At low [S]: velocity (v) increases proportionally (first-order kinetics).
• At high [S]: velocity plateaus at Vmax (zero-order kinetics) - enzyme is saturated.
• Relationship described by the Michaelis-Menten equation:

v = (Vmax × [S]) / (Km + [S])

2. Enzyme Concentration

• When substrate is in excess, velocity is directly proportional to enzyme concentration.
• Doubling enzyme doubles velocity.

3. Temperature

• Velocity increases with temperature up to an optimum (usually ~37°C in humans).
• Beyond the optimum: denaturation of enzyme protein occurs → velocity falls sharply.
• A 10°C rise in temperature roughly doubles enzyme activity (Q10 = 2) up to the optimum.

4. pH

• Each enzyme has an optimal pH at which activity is maximum.
  • Pepsin: pH 1-2 (acidic stomach)
  • Salivary amylase: pH 6.8-7.0
  • Trypsin: pH 8.0 (alkaline small intestine)
  • Arginase: pH 9.5-9.9
• Extreme pH causes denaturation and alters ionization of active site residues.

5. Product Concentration

• Accumulation of product inhibits the reaction by reverse reaction (product inhibition).
• Removal of product drives the reaction forward (Le Chatelier's principle).

6. Activators

• Some enzymes require metal ion activators (cofactors) for full activity.
  • Na⁺ activates plasma membrane ATPase
  • Mg²⁺ activates hexokinase and many kinases
  • Ca²⁺ activates some kinases
  • Cl⁻ activates salivary amylase

7. Inhibitors

• Substances that decrease enzyme velocity.
• Include competitive inhibitors, non-competitive inhibitors, uncompetitive inhibitors, and irreversible inhibitors (discussed in Q4).

8. Allosteric Effectors

• Regulatory molecules that bind at sites other than the active site.
• Positive effectors increase activity; negative effectors decrease it.

9. Coenzymes / Prosthetic Groups

• Many enzymes are inactive without their coenzyme. NAD⁺, FAD, CoA etc. are essential for activity.

10. Radiation and Chemical Agents

• UV radiation, heavy metals (Pb²⁺, Hg²⁺) inactivate enzymes by damaging protein structure or forming covalent bonds with -SH groups.

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Q4. Types of Enzyme Inhibition with Examples [5 Marks]

A. Reversible Inhibition

1. Competitive Inhibition

• The inhibitor (I) resembles the substrate structurally and competes for the same active site.
• It forms an enzyme-inhibitor (EI) complex instead of ES.
• Effect: Km is apparently increased (lower affinity). Vmax is unchanged (can be overcome by excess substrate).
• On Lineweaver-Burk plot: lines intersect on the Y-axis (same Vmax, different slope).
• Example: Malonate inhibits succinate dehydrogenase (resembles succinate). Statins inhibit HMG-CoA reductase. Allopurinol/Oxypurinol inhibits xanthine oxidase (see Q8).

2. Non-competitive Inhibition

• Inhibitor binds at a site other than the active site (allosteric site), either to free enzyme (E) or ES complex.
• Binding does not prevent substrate binding; Km is unchanged. But the ES complex cannot proceed to form product efficiently → Vmax is decreased.
• Cannot be overcome by adding more substrate.
• On Lineweaver-Burk plot: lines intersect on the X-axis (same Km, different Y-intercept).
• Example: Heavy metal ions (Hg²⁺, Pb²⁺) binding to -SH groups.

3. Uncompetitive Inhibition

• Inhibitor binds only to the ES complex, not the free enzyme.
• Both Km and Vmax are apparently decreased (in the same proportion).
• On Lineweaver-Burk plot: parallel lines (same slope, different intercepts).
• Example: Lithium inhibits inositol monophosphatase uncompetitively.

B. Irreversible Inhibition

• Inhibitor forms a covalent bond with the enzyme; activity cannot be recovered by dilution.
• Examples:
  • Organophosphates (nerve agents, insecticides like DFP) - irreversibly inhibit acetylcholinesterase by phosphorylating the serine residue at the active site.
  • Lead (Pb²⁺) - irreversibly inhibits ferrochelatase (involved in heme synthesis) by reacting with -SH (cysteine) groups.
  • Aspirin - irreversibly inhibits cyclooxygenase (COX) by acetylating serine residue.
  • Penicillin - irreversibly inhibits transpeptidase (involved in bacterial cell wall synthesis).

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Q5. Allosteric Enzymes [3 Marks]

Key Features:

1. Sigmoidal (S-shaped) curve when v₀ is plotted vs. [S] (not hyperbolic like M-M enzymes).
2. Regulated by allosteric effectors (modulators) which bind non-covalently at the allosteric site.
   • Positive effectors (activators): increase enzyme activity.
   • Negative effectors (inhibitors): decrease enzyme activity.
3. Homotropic effectors: When the substrate itself acts as an effector (cooperativity). Binding of one substrate molecule enhances binding at other subunits.
4. Heterotropic effectors: When the effector is a molecule different from the substrate (e.g., feedback inhibition by end-product).

Example:

• Aspartate transcarbamoylase (ATCase) - the paradigm allosteric enzyme. Inhibited by CTP (end product), activated by ATP.
• Phosphofructokinase-1 (PFK-1) in glycolysis - inhibited by citrate and ATP; activated by AMP and ADP.
• Hemoglobin behaves like an allosteric protein (O₂ is a homotropic effector; 2,3-BPG is a heterotropic effector).

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Q6. Regulation of Enzyme Activity [5 Marks]

1. Allosteric Regulation

• Effector molecules bind at the allosteric site and alter enzyme conformation → change in activity.
• Feedback inhibition is a classic example: the end product of a pathway inhibits the first committed enzyme (e.g., CTP inhibits ATCase).

2. Covalent Modification (Phosphorylation/Dephosphorylation)

• Protein kinases add phosphate groups (from ATP) to serine, threonine, or tyrosine residues.
• Phosphoprotein phosphatases remove phosphate groups.
• Phosphorylation can activate or inhibit an enzyme depending on the enzyme.
• Example: Glycogen phosphorylase is activated by phosphorylation; Glycogen synthase is inhibited by phosphorylation.

3. Zymogen (Proenzyme) Activation

• Enzymes are synthesized in an inactive precursor form (zymogen/proenzyme).
• Activated by irreversible proteolytic cleavage.
• Example: Pepsinogen → Pepsin (by HCl and pepsin itself). Trypsinogen → Trypsin (by enterokinase).
• Provides a mechanism to prevent autodigestion (e.g., pancreatic enzymes) and allows rapid activation when needed.

4. Induction and Repression of Enzyme Synthesis (Genetic Regulation)

• The amount of enzyme in the cell is regulated at the level of gene expression.
• Inducers increase transcription of the gene → more enzyme synthesized.
• Repressors decrease transcription → less enzyme.
• Example: Glucocorticoids induce synthesis of gluconeogenic enzymes (e.g., PEPCK).
• This is a slower form of regulation (hours to days) compared to allosteric changes (seconds).

5. Regulation by Hormones (Second Messenger Cascades)

• Hormones bind cell surface receptors → activate second messengers (cAMP, Ca²⁺) → activate protein kinases → phosphorylate target enzymes.
• Example: Epinephrine → ↑cAMP → activates Protein Kinase A → phosphorylates glycogen phosphorylase kinase → activates glycogen phosphorylase → glycogenolysis.

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Q7. Isoenzymes (Isozymes) - Definition + 2 Clinically Important Isoenzymes [5 Marks]

Definition

• Primary amino acid sequence
• Physical and chemical properties (electrophoretic mobility, heat stability, Km, Vmax, pH optimum)
• Tissue distribution
• They are products of different genes or of the same gene with different mRNA processing.

2 Clinically Important Isoenzymes:

1. Creatine Kinase (CK) / Creatine Phosphokinase (CPK)

• Reaction: Creatine + ATP ⇌ Creatine phosphate + ADP
• CK is a dimer made of two subunits: M (muscle) and B (brain).
• Three isoforms:

  Isoenzyme	Composition	Location
  CK-MM	MM	Skeletal muscle (96% of normal serum CK)
  CK-MB	MB	Heart muscle (cardiac specific)
  CK-BB	BB	Brain, smooth muscle, lung

• Clinical significance: CK-MB rises 4-8 hours after myocardial infarction (MI), peaks at 24 hours, and returns to normal by 48-72 hours. CK-MB > 5% of total CK is diagnostic of MI.

2. Lactate Dehydrogenase (LDH)

• Reaction: Pyruvate + NADH ⇌ Lactate + NAD⁺
• LDH is a tetramer of two subunits: H (heart) and M (muscle).
• Five isoforms:

  Isoenzyme	Composition	Location
  LDH-1	HHHH	Heart, RBCs
  LDH-2	HHHM	Heart, RBCs
  LDH-3	HHMM	Lung, spleen
  LDH-4	HMMM	Kidney, liver
  LDH-5	MMMM	Liver, skeletal muscle

• Normal serum: LDH-2 > LDH-1.
• In MI: LDH-1 > LDH-2 (called the "LDH flip") - appears 12-24 hrs after MI, peaks at 48-72 hrs, remains elevated for 10-14 days.
• Used historically when CK-MB had returned to normal (late presentation).

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Q8. Allopurinol in Gout - Type of Inhibition + Mechanism [5 Marks]

Type of Inhibition

Background - Gout and Uric Acid

• Gout is a disorder of purine metabolism resulting in hyperuricemia (elevated serum uric acid).
• Uric acid (the final product of purine catabolism in humans) precipitates as monosodium urate crystals in joints, causing acute arthritis.

Mechanism of Allopurinol

Hypoxanthine → Xanthine → Uric acid

1. Allopurinol (a structural analog of hypoxanthine) initially acts as a competitive inhibitor by binding to the active site of xanthine oxidase.
2. Xanthine oxidase then oxidizes allopurinol to oxypurinol (alloxanthine) - allopurinol is the "suicide substrate."
3. Oxypurinol remains tightly bound to the reduced (Mo⁴⁺) form of xanthine oxidase at the active site, resulting in irreversible/tight-binding inhibition (mechanism-based inhibition).
4. This inhibition of xanthine oxidase prevents the conversion of hypoxanthine and xanthine to uric acid.
5. Hypoxanthine and xanthine (which are more water-soluble than uric acid) are excreted in urine instead.
6. Result: Serum uric acid levels fall → relief of gout.

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Q9. Active Site of an Enzyme - Definition + Characteristics [3 Marks]

Definition

Characteristics:

1. Small and specific: Occupies only a small fraction (1-2%) of the total enzyme surface, yet it determines the substrate specificity of the enzyme.
2. Three-dimensional structure: The active site is formed by amino acid residues that may be far apart in the primary sequence but come together due to protein folding.
3. Non-polar (hydrophobic) microenvironment: Most of the active site is hydrophobic, which favors substrate binding and catalysis.
4. Contains two types of sites:
   • Binding site - holds the substrate in the correct orientation.
   • Catalytic site - amino acid residues that directly participate in the chemical reaction.
5. Flexible - not rigid: The active site can change shape upon substrate binding (as per the induced fit model).
6. Complementary to the substrate: The shape, charge, and chemical properties are complementary to the substrate (like a lock and key).
7. Amino acids commonly involved: Serine (Ser), Histidine (His), Aspartate (Asp), Cysteine (Cys), Lysine (Lys), and sometimes metal ions.
8. Water molecules at the active site may also be displaced upon substrate binding, contributing to binding energy.

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Q10. Theories Proposed for Enzyme-Substrate Binding [5 Marks]

1. Lock and Key Model (Fischer's Theory, 1894)

• Proposed by Emil Fischer.
• The enzyme active site has a rigid, preformed shape that is exactly complementary to the substrate.
• The substrate fits into the active site like a key fits into a lock - perfect geometric complementarity.
• Limitation: This model cannot explain:
  • How enzymes can catalyze the reverse reaction.
  • Why some enzymes can act on structurally similar but different substrates.
  • The observed conformational changes in enzymes upon substrate binding.
  • Why the transition state (not just substrate) is stabilized.

2. Induced Fit Model (Koshland's Theory, 1958)

• Proposed by Daniel Koshland.
• The active site is not rigid but flexible.
• When the substrate approaches and binds to the enzyme, it induces a conformational change in the active site, bringing catalytic residues into proper alignment.
• The enzyme "molds" itself around the substrate.
• This model explains:
  • Why substrate binding increases enzyme activity (proper alignment of catalytic groups occurs only after substrate binding).
  • Negative cooperativity - binding of one substrate can decrease affinity at another site.
  • Broad substrate specificity of some enzymes.
  • The stabilization of the transition state.
• Currently accepted as the more accurate model.
• Example: Hexokinase undergoes a large conformational change upon binding glucose, excluding water from the active site and preventing futile hydrolysis of ATP.

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Q11. Km of an Enzyme - Definition and Significance [3 Marks]

Definition

v = (Vmax × [S]) / (Km + [S])

Km = [S] at Vmax/2

Significance of Km:

1. Measure of affinity: Km reflects the affinity of the enzyme for its substrate.
   • Low Km = high affinity (enzyme needs less substrate to achieve half-maximal velocity).
   • High Km = low affinity (more substrate needed).
2. Km is a constant for a given enzyme-substrate pair under fixed conditions of pH and temperature.
3. Comparison of substrates: When an enzyme has multiple substrates, the one with the lowest Km is the preferred (physiological) substrate.
4. Indicator of in vivo substrate saturation: If intracellular [S] >> Km, the enzyme is nearly saturated and works at near-Vmax. If [S] ~ Km, the enzyme is sensitive to changes in substrate concentration.
5. Clinical use: In competitive inhibition, the apparent Km increases (Vmax unchanged). In non-competitive inhibition, Km is unchanged (Vmax decreases). This helps identify the type of inhibition.
6. Example: Hexokinase (Km for glucose ~0.1 mM) vs. Glucokinase (Km ~10 mM) - explains why glucokinase only acts on glucose after a meal when glucose is high.

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Q12. Types of Enzyme Specificities [5 Marks]

1. Absolute Specificity

• The enzyme catalyzes only one specific reaction with one specific substrate.
• Most strict form of specificity.
• Example: Urease acts only on urea. Succinic dehydrogenase acts only on succinate.

2. Group Specificity (Relative Specificity)

• The enzyme acts on molecules with a specific chemical group or bond, regardless of the rest of the molecule.
• Example:
  • Hexokinase phosphorylates many hexoses (glucose, fructose, mannose) - it requires the 6-OH group.
  • Amino acid oxidase acts on any L-amino acid.

3. Linkage (Bond) Specificity

• The enzyme acts on a particular type of chemical bond regardless of the molecular structure around it.
• Example:
  • Esterases hydrolyze ester bonds (-COO-) in various esters.
  • Proteases (non-specific) cleave peptide bonds.

4. Stereochemical Specificity (Stereospecificity)

• Enzymes can distinguish between stereoisomers (optical isomers and geometric isomers).
• a) Optical (chiral) specificity:
  • L-amino acid oxidase acts only on L-amino acids (not D-amino acids).
  • D-amino acid oxidase acts only on D-amino acids.
• b) Geometric isomer specificity:
  • Fumarase catalyzes hydration of fumarate (trans form) but not maleate (cis form).
• This is because the enzyme active site is asymmetric (chiral environment).

5. Reaction Specificity

• Each enzyme catalyzes only one type of reaction with a given substrate.
• Example: Glucose-6-phosphate can be acted upon by different enzymes for different reactions - glucose-6-phosphatase (hydrolysis), glucose-6-phosphate dehydrogenase (oxidation), phosphoglucomutase (isomerization).

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Q13. Therapeutic Applications of Enzymes [3/5 Marks]

A. Enzymes Used as Drugs (Therapeutic Enzymes)

B. Enzymes Used as Reagents in Diagnosis (Mentioned under therapeutic/diagnostic use)

• Enzymes are also used to assay metabolites (e.g., glucose oxidase to measure serum glucose).

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Q14. Enzyme Profile in Myocardial Infarction (MI) [5 Marks]

Key Enzymes:

Diagnostic Sequence:

1. First hours (0-8h): Total CK and CK-MB begin to rise; Troponins also rise.
2. 24 hours: CK-MB peaks - best time to confirm MI.
3. Late diagnosis (2-3 days after): CK-MB may have normalized; LDH-1 is still elevated - LDH flip (LDH1 > LDH2) is diagnostic.
4. Up to 10 days: Troponins remain elevated for the longest period, allowing late diagnosis.

Clinical Note:

• Cardiac Troponins I and T (cTnI, cTnT) are the current "gold standard" for diagnosing MI.
• They appear in plasma within 4-6 hours, peak at 24-36 hours, and remain elevated for 3-10 days.
• The ratio of CK-MB/Total CK > 5% (or CK index > 5%) is considered diagnostic of MI.
• LDH flip (LDH1 > LDH2) is seen in: MI, megaloblastic anemia, hemolytic anemia.

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Q15. Coenzymes - Definition + Role in Metabolism with 2 Examples [3 Marks]

Definition

• If tightly and covalently associated: called a prosthetic group.
• Enzyme without coenzyme = apoenzyme (inactive).
• Enzyme + coenzyme = holoenzyme (active).

Role in Metabolism with 2 Examples:

1. NAD⁺ (Nicotinamide Adenine Dinucleotide) - derived from Niacin (Vitamin B₃)

• Acts as an electron carrier / hydrogen acceptor in oxidation-reduction reactions.
• Accepts 2 electrons and 1 proton from the substrate: NAD⁺ + 2H → NADH + H⁺
• Role: Accepts hydride ions (H⁻) from substrates being oxidized in:
  • Glycolysis (glyceraldehyde-3-phosphate dehydrogenase)
  • TCA cycle (isocitrate DH, α-ketoglutarate DH, malate DH)
  • Beta-oxidation of fatty acids
• NADH then donates electrons to the electron transport chain for ATP synthesis.

2. Coenzyme A (CoA) - derived from Pantothenic Acid (Vitamin B₅)

• Acts as a carrier of acyl groups (thiol ester bond).
• The reactive -SH (thiol) group of CoA forms thioester bonds with acyl groups.
• Role:
  • Acetyl CoA is the entry point into the TCA cycle.
  • Fatty acyl-CoA is the activated form for beta-oxidation.
  • Malonyl CoA is the building block for fatty acid synthesis.
  • Succinyl CoA is an intermediate in heme synthesis and the TCA cycle.

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Q16. Proenzymes (Zymogens) - Definition + 2 Examples [3 Marks]

Definition

1. Prevent unwanted enzymatic activity in the cell of synthesis (e.g., autodigestion of the pancreas).
2. Allow rapid activation when and where needed.

2 Examples:

1. Pepsinogen → Pepsin

• Pepsinogen (MW ~42,000) is secreted by chief cells (peptic cells) of the gastric mucosa.
• Activated by HCl (gastric acid) and by pepsin itself (autocatalysis/autoactivation).
• Mechanism: A 44-amino acid N-terminal peptide is cleaved, exposing the active site of pepsin.
• Pepsin (MW ~35,000) is an endopeptidase that cleaves proteins at peptide bonds involving aromatic amino acids (Phe, Tyr, Trp).

2. Trypsinogen → Trypsin

• Trypsinogen is secreted by the exocrine pancreas.
• Activated by enterokinase (enteropeptidase) secreted by the duodenal mucosa.
• Mechanism: Enterokinase cleaves a specific hexapeptide from the N-terminus of trypsinogen, generating trypsin.
• Trypsin then activates other pancreatic zymogens: chymotrypsinogen, proelastase, procarboxypeptidase, prolipase - in a cascade activation.
• Clinical note: In acute pancreatitis, premature activation of trypsinogen inside the pancreas leads to self-digestion.

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Q17. 5 Enzymes of Diagnostic Significance with Normal Serum Levels [5 Marks]

(Note: At least 5 required for full marks - the table above provides extra for completeness. The 5 most commonly asked are: ALT, AST, ALP, CK-MB, and LDH.)

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