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Question 2: Factors Affecting Enzyme Activity + Properties of Enzymes

Properties of Enzymes

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Factors Affecting Enzyme Activity

1. Temperature

• Every 10°C rise in temperature approximately doubles enzyme activity (Q10 effect).
• However, high temperatures cause denaturation — enzymes lose their 3D shape and activity.
  • Example: Creatine kinase (CK) denatures at 37°C; amylase at 45°C.
• Exception: Taq polymerase remains stable at 95°C (used in PCR).
• Low temperatures slow reactions but preserve enzyme stability — useful for storing samples.

2. pH

• Each enzyme has an optimal pH at which it works best.
  • Pepsin (stomach): optimal pH ~2 (acidic)
  • Trypsin (small intestine): optimal pH ~8 (slightly alkaline)
  • Most body enzymes: optimal pH ~7.4
• pH affects the ionization of amino acids in the active site, changing enzyme shape and substrate binding. Extreme pH causes denaturation.

3. Substrate Concentration ([S])

• As [S] increases, reaction velocity (v) increases — more enzyme-substrate complexes form.
• At very high [S], the enzyme becomes saturated and velocity reaches a maximum (Vmax).
• This relationship follows Michaelis-Menten kinetics: the Km (Michaelis constant) is the [S] at which velocity = ½ Vmax. A lower Km means higher affinity for the substrate.

4. Enzyme Concentration

• If substrate is in excess, reaction rate is directly proportional to enzyme concentration — more enzyme molecules = more active sites = faster reaction.

5. Cofactors and Coenzymes

• Many enzymes require non-protein helpers:
  • Cofactors: inorganic ions (Mg²⁺, Zn²⁺, Fe²⁺)
  • Coenzymes: organic molecules (NAD⁺, FAD, Coenzyme A — most derived from vitamins)

6. Inhibitors (see Question 3 below)

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Question 3: Enzyme Inhibition — Definition and Types

Definition

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Types of Enzyme Inhibition

A. Irreversible Inhibition

• Inhibitor binds to the enzyme via a covalent bond — permanent, cannot be reversed by dilution.
• Destroys enzyme function permanently.
• Example: Lead (Pb²⁺) irreversibly inhibits ferrochelatase (an enzyme in heme synthesis) by forming covalent bonds with cysteine's –SH group.
• Aspirin irreversibly inhibits cyclooxygenase (COX), blocking prostaglandin synthesis.

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B. Reversible Inhibition

1. Competitive Inhibition

• The inhibitor looks like the substrate and competes for the same active site.
• Substrate and inhibitor cannot bind simultaneously.

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2. Noncompetitive Inhibition

• The inhibitor binds to a different site (allosteric site) — not the active site.
• Can bind to free enzyme OR the enzyme-substrate (ES) complex.
• Substrate binding is unaffected, but the reaction cannot proceed normally.

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3. Uncompetitive Inhibition

• Inhibitor binds only to the ES complex (not free enzyme).
• Both Vmax and Km are decreased proportionally.
• Lineweaver-Burk plot: Parallel lines (same slope, different intercepts).

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Quick Comparison Table

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Drug Examples (Important for Pharm D)

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Factors Affecting Enzyme Activity + Properties of Enzymes

(Pharm D 1st Year — Biochemistry)

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PART 1: FACTORS AFFECTING ENZYME ACTIVITY

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1. 🌡️ Temperature

• Enzyme activity increases as temperature rises — generally, every 10°C increase doubles the reaction rate (Q₁₀ effect).
• This is because higher temperature increases molecular kinetic energy, leading to more frequent enzyme-substrate collisions.
• However, above an optimal temperature, the enzyme begins to denature — its 3D structure unfolds, active site is destroyed, and activity drops sharply to zero.

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2. 🧪 pH

• Every enzyme has an optimum pH at which it shows maximum activity.
• Changes in pH affect the ionization of amino acid residues in the active site, altering the enzyme's shape and ability to bind substrate.
• Extreme pH values on either side cause denaturation.

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3. 📊 Substrate Concentration [S]

• As substrate concentration increases, reaction velocity (v) increases — more substrates fill available active sites.
• At very high [S], all active sites are occupied → the enzyme is saturated → velocity reaches a maximum (Vmax).
• This follows Michaelis-Menten kinetics:

• Km (Michaelis constant) = the [S] at which v = ½ Vmax
  • Low Km = high affinity for substrate (less substrate needed)
  • High Km = low affinity for substrate

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4. ⚗️ Enzyme Concentration [E]

• When substrate is in excess, reaction rate is directly proportional to enzyme concentration.
• More enzyme molecules = more active sites available = faster product formation.
• Clinically, enzyme concentration in blood is measured to diagnose disease (e.g., elevated ALT/AST in liver damage).

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5. 🔩 Cofactors and Coenzymes (Activators)

• The complete, active enzyme (protein + cofactor/coenzyme) = Holoenzyme
• The inactive protein part alone = Apoenzyme
• Most coenzymes are derived from B-vitamins — this is why B-vitamin deficiency causes metabolic disease.

Holoenzyme = Apoenzyme + Cofactor/Coenzyme

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6. 🚫 Inhibitors

• Irreversible — covalent binding (e.g., aspirin, lead)
• Reversible — competitive, noncompetitive, uncompetitive

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7. 🧂 Ionic Strength / Salt Concentration

• High ionic strength decreases enzyme activity by disrupting the ionic interactions that stabilize enzyme structure.
• Dilution of clinical samples with appropriate enzyme diluents (containing plasma proteins like albumin) is necessary to maintain accurate enzyme measurements.

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8. ⚡ Allosteric Regulation

• Small molecules called allosteric effectors bind to a site other than the active site.
• They cause conformational changes in the enzyme:
  • Allosteric activators → increase activity
  • Allosteric inhibitors → decrease activity
• This is the main mechanism for feedback regulation in metabolic pathways.

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PART 2: NOTE ON PROPERTIES OF ENZYMES

What Are Enzymes?

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Key Properties

1. Proteins in Nature

2. Catalytic Function

• Enzymes speed up reactions by lowering the activation energy (Ea) — the energy needed to start the reaction.
• They do NOT change the equilibrium of a reaction (the final ratio of products to reactants is unchanged — only the speed of reaching equilibrium changes).

3. High Specificity

• Binding specificity: Each enzyme binds only one or a few specific substrates.
• Reaction specificity: Each enzyme catalyzes only one type of chemical reaction — virtually no by-products are formed.
• Stereospecificity: Enzymes recognize only one mirror-image (enantiomeric) form of a substrate. Example: Proteases act only on L-amino acids (not D-amino acids).

4. Active Site

• The active site is a small region on the enzyme where the substrate binds and the reaction occurs.
• Two models explain enzyme-substrate binding:
  • Lock and Key model: Active site is a rigid, pre-formed shape that perfectly fits the substrate.
  • Induced Fit model (more accepted): Active site is flexible; it changes shape when the substrate binds to achieve the best fit.

5. Not Consumed in Reaction

• Enzymes are regenerated after each reaction cycle — one enzyme molecule can process thousands of substrate molecules per second.

6. Regulated

• Enzyme activity can be turned up or down based on cellular needs via:
  • Allosteric regulators
  • Covalent modification (e.g., phosphorylation)
  • Feedback inhibition (product inhibits early enzyme in a pathway)
  • Induction/repression of gene expression

7. Cellular Compartmentalization

• Enzymes are localized in specific organelles. Example:
  • Krebs cycle enzymes → mitochondria
  • Digestive enzymes → lysosomes
  • Glycolytic enzymes → cytoplasm
• This isolates reactions, prevents interference, and increases efficiency.

8. Cofactor Requirement

• Some enzymes need non-protein helpers (cofactors or coenzymes) for activity — without them, they are inactive (apoenzyme form).

9. Enzyme Specificity as Biomarkers

• Because many enzymes are tissue-specific, elevated levels in blood indicate organ damage.
  • Elevated ALT/AST → liver disease
  • Elevated CK-MB → myocardial infarction (heart attack)
  • Elevated amylase/lipase → pancreatitis

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Summary Table