Enzymes: Chemistry, Classification, Kinetics, Inhibition, and Mechanism of Action

Course Information

• Course Name & Code: Pharmaceutical Biochemistry-I (PHL-309)
• Topic: Enzymes: Chemistry, Classification, Kinetics, Inhibition, and Mechanism of Action
• Credit Hours: 3

Learning Objectives

By the end of this topic, students will be able to:

• Define enzymes and explain their chemical nature and classification.
• Describe the mechanism of enzyme action, including the concepts of active site, substrate specificity, and transition state stabilization.
• Understand enzyme kinetics, including the Michaelis‑Menten equation, its modifications, and factors affecting reaction rates.
• Differentiate between types of enzyme inhibition and their clinical significance.
• Explain the regulation of allosteric enzymes and their role in metabolic control.
• Discuss the biological and pharmaceutical importance of enzymes.
• Describe the mechanism of action of chymotrypsin and ribonuclease.

Core Content

3.1 Introduction to Enzymes

Definition

Enzymes are biological catalysts—typically proteins (with the exception of some catalytic RNA molecules called ribozymes)—that increase the rate of chemical reactions in living organisms without being consumed or permanently altered in the process.

Key Characteristics

• Specificity: Enzymes are highly specific for their substrates.
• Efficiency: Enzymes accelerate reaction rates by factors of 10⁶ to 10¹² compared to uncatalyzed reactions.
• Regulation: Enzyme activity is tightly controlled to meet cellular needs.
• Mild Conditions: Enzymes function under physiological conditions (temperature ~37°C, pH ~7.4).

Historical Background

• 1833: Payen and Persoz discovered the first enzyme, diastase (amylase).
• 1878: Kühne coined the term "enzyme" (from Greek: "in yeast").
• 1894: Emil Fischer proposed the "Lock and Key" model of enzyme-substrate specificity.
• 1926: Sumner first crystallized urease and proved enzymes are proteins (Nobel Prize in 1946).
• 1948: Pauling proposed the concept of transition state stabilization.
• 1958: Koshland proposed the "Induced Fit" model.

Chemical Nature of Enzymes

Structure of Enzymes

• Active Site: A small region (3‑15 amino acids) where substrate binding and catalysis occur.
  • Contains catalytic residues that participate in bond breaking/forming.
  • Often contains metal ions for catalytic function.
• Allosteric Site: A distinct site where regulatory molecules (modulators) bind.
  • Binding induces conformational changes that increase or decrease activity.
• Substrate Binding Sites:
  • Lock and Key Model (Fischer, 1894): The active site has a rigid shape exactly complementary to the substrate.
  • Induced Fit Model (Koshland, 1958): The active site changes conformation upon substrate binding to achieve optimal fit.
• Transition State Stabilization:
  • Enzymes stabilize the high-energy transition state, lowering the activation energy (ΔG°‡).
  • The enzyme does not change the equilibrium constant (Kₑq) but dramatically increases the rate of reaching equilibrium.

3.2 Classification of Enzymes

The Enzyme Commission (EC) of the IUBMB classifies enzymes into 6 major classes based on the type of reaction they catalyze:

3.3 Enzyme Specificity

3.4 Mechanism of Enzyme Action

General Steps

1. Substrate Binding: Substrate (S) binds to the active site, forming an enzyme-substrate complex (ES).
2. Transition State Formation: The enzyme stabilizes the transition state, lowering activation energy.
3. Catalysis: Chemical bonds are made/broken; product (P) is formed.
4. Product Release: Product is released, and the enzyme is regenerated.

Equation: E + S ⇌ ES → E + P

Factors Contributing to Enzyme Catalysis

3.5 Enzyme Kinetics

3.5.1 Michaelis-Menten Equation

Definition: The Michaelis-Menten equation describes the relationship between substrate concentration [S] and reaction velocity (V).

Assumptions:

• Single substrate reaction (or no inhibition).
• Steady-state assumption: [ES] is constant (d[ES]/dt = 0).
• Total enzyme concentration [E]ₜ is constant.
• Reaction is irreversible (at least initially).

Derivation:
E + S ⇌ (k₁, k₋₁) ES → (k₂) E + P

Rate Equations:

• Rate of formation of ES: k₁[E][S] + k₋₂[E][P]
• Rate of breakdown of ES: k₋₁[ES] + k₂[ES]

Steady-State (simplified):
k₁[E][S] = (k₋₁ + k₂)[ES]

Michaelis-Menten Equation:
V₀ = (V_max × [S]) / (K_m + [S])

where:

• V₀ = Initial velocity
• V_max = Maximum velocity (at saturating [S])
• [S] = Substrate concentration
• K_m = Michaelis constant = (k₋₁ + k₂)/k₁

3.5.2 Lineweaver-Burk Plot (Double Reciprocal Plot)

Double reciprocal equation:
1/V₀ = (K_m/V_max) × (1/[S]) + 1/V_max

Plot: 1/V₀ (y-axis) vs 1/[S] (x-axis) → a straight line.

Advantages:

• Easy to determine V_max and K_m.
• Useful for identifying types of enzyme inhibition.

3.6 Enzyme Inhibition

Definition: Enzyme inhibition is the decrease in enzyme activity caused by inhibitors (molecules that bind to the enzyme and reduce its catalytic activity).

3.6.1 Types of Inhibition

A. Reversible Inhibition (Inhibitor binds non-covalently; activity can be restored)

Competitive Inhibition Details:

• Overcome by high substrate concentration ([S]).
• Examples: Statins (HMG-CoA reductase), Methotrexate (dihydrofolate reductase), Sulfonamides (p-aminobenzoic acid analogues), Malonate (succinate dehydrogenase).

Non-Competitive Inhibition Details:

• Cannot be overcome by high [S].
• Examples: Heavy metal ions (Pb²⁺, Hg²⁺) binding to sulfhydryl groups; DIPF (diisopropyl fluorophosphate) irreversibly inhibits serine proteases.

Uncompetitive Inhibition Details:

• Rare; inhibitor only binds to ES complex.
• Examples: Some inhibitors of alkaline phosphatase; Lithium (in some reactions).

B. Irreversible Inhibition

• Inhibitor covalently binds to enzymes (permanent inactivation).
• Examples:
  • Aspirin (Acetylsalicylic Acid): irreversibly acetylates cyclooxygenase (COX) → inhibits prostaglandin synthesis.
  • Penicillin: irreversibly inhibits bacterial transpeptidase (cell wall synthesis).
  • Organophosphates (Sarin, Malathion): irreversibly inhibit acetylcholinesterase (→ nerve gas poisoning).
  • DIPF: inhibits serine proteases.

3.6.2 Clinical and Pharmaceutical Importance of Enzyme Inhibition

3.7 Factors Affecting Enzyme Activity

3.7.1 Substrate Concentration ([S])

• As [S] increases, V₀ increases until V_max is reached (saturation).
• Equation: V₀ = (V_max × [S]) / (K_m + [S])

3.7.2 Enzyme Concentration ([E])

• Proportionality: V₀ ∝ [E] (under substrate saturating conditions).
• This is the basis for enzyme assays and quantification.

3.7.3 Temperature

• Optimum Temperature: Most mammalian enzymes ~37°C.
• Effect of Heat:
  • Below optimum: V increases (more kinetic energy).
  • At optimum: Peak activity.
  • Above optimum: Denaturation (loss of tertiary/quaternary structure).
• Temperature Coefficient (Q₁₀): For most enzymes, V doubles for every 10°C increase.

3.7.4 pH

• Optimum pH: Varies per enzyme (e.g., pepsin pH 1.5, trypsin pH 8.0, alkaline phosphatase pH 9-10).
• Effect: Changes in ionization of active site residues (histidine, cysteine, lysine) affect substrate binding and catalysis.
• Enzymes have a narrow pH range (denature at extremes).

3.7.5 Activators and Inhibitors

• Activators: Ions (Mg²⁺, Ca²⁺, Zn²⁺) or small molecules that increase activity.
• Inhibitors: As described above (competitive, non-competitive, irreversible).

3.7.6 Effect of Product Accumulation

• Feedback inhibition: product accumulation inhibits the first enzyme in a pathway.

3.8 Allosteric Enzymes

Definition

Allosteric enzymes are regulatory enzymes that have binding sites for effector molecules (allosteric modulators) distinct from the active site. Binding of effectors induces conformational changes that alter enzyme activity.

Characteristics

Kinetic Behavior

• Sigmoidal Kinetics: The curve of V₀ vs [S] is S-shaped.
  • At low [S]: binding is less cooperative (low affinity).
  • At high [S]: cooperativity increases (high affinity).
• Significance: Sigmoidal kinetics allows a rapid, switch-like response to changes in substrate or effector concentrations.

Types of Allosteric Modulators

Clinical/Pharmaceutical Importance of Allosteric Enzymes

Models of Allosteric Regulation

1. Concerted Model (Monod-Wyman-Changeux, MWC Model):

• All subunits exist in either R (Relaxed, active) or T (Tensed, inactive) states.
• All subunits switch simultaneously (concerted).
• Substrate binds preferentially to R state.
• Effectors:
  • Positive: Stabilize R state.
  • Negative: Stabilize T state.

2. Sequential Model (Koshland-Némethy-Filmer, KNF Model):

• Subunits change conformation individually upon ligand binding.
• Conformational changes propagate to neighboring subunits.
• Cooperativity arises from progressive conformational changes.

3.9 Biological Importance of Enzymes

3.10 Pharmaceutical Importance of Enzymes

3.11 Mechanism of Action of Important Enzymes

3.11.1 Chymotrypsin

Definition: Chymotrypsin is a serine protease (EC 3.4.21.1) that hydrolyzes peptide bonds adjacent to hydrophobic amino acids (Phe, Tyr, Trp, Leu, Met). Source: Pancreatic secretions; synthesized as an inactive precursor (zymogen) chymotrypsinogen, activated by trypsin.

Structure:

• Single polypeptide chain (245 amino acids) with a catalytic triad.
• Catalytic Triad: Ser195, His57, Asp102.
• Oxyanion hole stabilizes the transition state.

Mechanism of Action (Covalent Catalysis - Two-step Mechanism):

Step 1: Acylation:

1. Binding: Substrate binds to the active site; hydrophobic pocket accommodates the aromatic/large hydrophobic side chain.
2. Nucleophilic Attack: Ser195 Oγ attacks the peptide carbonyl carbon.
3. Transition State Stabilization: Oxyanion hole forms hydrogen bonds with the carbonyl oxygen.
4. Hydrolysis: His57 abstracts proton from Ser195; tetrahedral intermediate forms.
5. Covalent Bond Formation: Ser195 acylates the substrate (acyl-enzyme intermediate).
6. Amino Group Release: The new N-terminus of the amino fragment is released via deacylation.

Step 2: Deacylation:

1. Water Molecule: Water enters the active site.
2. Hydrolysis: His57 deprotonates water; water attacks the acyl-enzyme intermediate.
3. Release: The acyl group is released as the carboxylic acid fragment; enzyme is regenerated.

Key Interactions:

• Specificity Pocket: Deep, lined with hydrophobic residues (Gly216, Ser189, Asp189) that accommodate aromatic/hydrophobic side chains.
• Oxyanion Hole: Stabilizes the transition state by hydrogen bonding.
• Catalytic Triad: Asp102 stabilizes His57, allowing it to abstract/give protons.

Substrates: Peptides with aromatic (Phe, Tyr, Trp) or bulky hydrophobic (Leu, Met) amino acids.

Inhibitors:

• Irreversible: DIPF (diisopropyl fluorophosphate) covalently binds to Ser195 (suicide inhibitor).
• Reversible: Aprotinin (pancreatic trypsin inhibitor), TPCK (specific for chymotrypsin).
• Clinical: Protease inhibitors are used for pancreatitis, bleeding disorders.

3.11.2 Ribonuclease (RNase A)

Definition: Ribonuclease A (RNase A, EC 4.6.1.19) is an endonuclease that cleaves single-stranded RNA by hydrolyzing the phosphodiester bond adjacent to pyrimidine nucleotides (cytosine, uracil). Source: Bovine pancreas.

Structure:

• Single polypeptide chain (124 amino acids) with four disulfide bonds.
• Catalytic Residues: His12, His119, Lys41 (critical for catalysis).
• Pyrimidine-specific binding pocket: Contains Thr45, Phe120, Ser123.

Mechanism of Action (Acid-Base Catalysis - Two-step Mechanism):

Step 1: Transphosphorylation (Cleavage of RNA chain):

1. Substrate Binding: RNA binds; pyrimidine base (U or C) is positioned in the specificity pocket.
2. Nucleophilic Attack: 2'-OH of ribose (immediately adjacent to the phosphate) attacks the phosphorus atom.
3. Acid-Base Catalysis: His12 acts as a base (abstracts proton from 2'-OH); His119 acts as an acid (donates proton to 5'-O leaving group).
4. Pentavalent Intermediate: Trigonal bipyramidal transition state.
5. Cleavage: 2',3'-cyclic phosphate intermediate is formed; 5'-OH product is released.

Step 2: Hydrolysis (Degradation of cyclic phosphate):

1. Water Molecule: Water enters the active site.
2. Nucleophilic Attack: Water (activated by His119) attacks the cyclic phosphate.
3. Ring Opening: 2',3'-cyclic phosphate is opened to yield 3'-phosphate end.
4. Release: 3'-phosphate nucleotide is released; enzyme is regenerated.

Key Interactions:

• Specificity Pocket: Binds pyrimidine bases (C and U) via hydrogen bonds (Thr45, Phe120, Ser123).
• Catalytic Residues: His12 and His119 carry out acid-base catalysis.
• Lys41: Stabilizes the phosphate group via electrostatic interactions.
• Disulfide Bonds: Stabilize the enzyme structure (RNase A has four disulfide bonds).

Substrates: Single-stranded RNA; cleaves after pyrimidine nucleotides (U or C).

Inhibitors:

• Non-specific: Heavy metal ions, ribonuclease inhibitor (protein).
• Specific: Uridine 2',3'-cyclic phosphate (transition state analogue).

Significance of RNase A:

• Classical Protein Folding Model: RNase A was one of the first proteins to be refolded in vitro (Anfinsen's experiment; Nobel Prize 1972).
• Catalytic Mechanism: A textbook example of acid-base catalysis.
• Pharmaceutical Relevance: RNases are being studied as anti-cancer agents (angiogenin).

3.12 Enzyme Regulation

4. Diagrams/Tables

Table 1: Classification of Enzymes (EC System)

Table 2: Comparison of Enzyme Inhibition Types

Table 3: Clinical Enzymes in Diagnosis

Table 4: Allosteric Regulation of Key Metabolic Enzymes

5. Exam Angle

Short Questions (2-5 marks)

1. Define enzymes and classify them according to the EC system.
2. What is K_m? Explain its significance.
3. Differentiate between competitive and non-competitive inhibition.
4. What are allosteric enzymes? Give examples.
5. Explain the catalytic triad of chymotrypsin.
6. What is the significance of the Lineweaver-Burk plot?
7. Define isozymes with examples.
8. What are the factors affecting enzyme activity?
9. Explain the induced-fit model of enzyme action.
10. Give the pharmaceutical importance of enzyme inhibitors with examples.

Long Essays (10+ marks)

1. Describe the mechanism of enzyme action. Discuss the factors affecting enzyme activity and their importance in pharmacology.
2. Derive the Michaelis-Menten equation and explain its application in enzyme kinetics. Add a note on enzyme inhibition.
3. Write a detailed note on allosteric enzymes, their regulation, and clinical significance.
4. Explain the mechanism of action of chymotrypsin and ribonuclease. Add a note on the pharmaceutical importance of serine proteases.
5. Discuss the biological and pharmaceutical importance of enzymes with suitable examples.

Key Points Frequently Tested

• Michaelis-Menten: V₀ = (V_max × [S]) / (K_m + [S]); K_m = affinity; V_max = [E]ₜ × k_cat.
• Inhibitors: Competitive (↑K_m, V_max unchanged), Non-competitive (↓V_max, K_m unchanged), Uncompetitive (↓both), Irreversible (permanent inhibition).
• Allosteric Enzymes: Sigmoidal kinetics, regulatory sites, feedback inhibition (e.g., PFK-1, HMG-CoA reductase).
• Clinical Enzymes: CK-MB (cardiac), ALT/AST (liver), ALP (bone/liver), Amylase/Lipase (pancreas).
• Catalytic Mechanisms: Chymotrypsin (covalent catalysis, catalytic triad Ser-His-Asp), RNase (acid-base catalysis, His12/His119).
• Drug Targets: Statins (HMG-CoA reductase), ACE inhibitors (ACE), Penicillin (transpeptidase), Aspirin (COX), Methotrexate (DHFR).

6. Summary Box

• Enzymes are protein catalysts that accelerate biochemical reactions with high specificity and efficiency.
• Enzyme classification (EC system): 6 major classes—oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.
• Michaelis-Menten kinetics describes the relationship between substrate concentration and reaction velocity (V₀ = V_max[S]/(K_m+[S])); K_m indicates affinity.
• Enzyme inhibition: Competitive (↑K_m), non-competitive (↓V_max), uncompetitive (↓both), and irreversible (permanent; drugs like aspirin, penicillin).
• Allosteric enzymes have regulatory sites; they display sigmoidal kinetics and are regulated by modulators (activators/inhibitors) that control metabolic pathways.
• Enzymes are vital in metabolism, DNA replication, signal transduction, and pharmaceutical drug action and diagnosis.
• Chymotrypsin is a serine protease with a catalytic triad (Ser-His-Asp) that uses covalent and acid-base catalysis. Ribonuclease A uses acid-base catalysis with His12 and His119 to cleave RNA at pyrimidine nucleotides.

7. References

• Chatterjee, M. N., & Shinde, R. (2011). Textbook of Medical Biochemistry (8th ed.). Jaypee Brothers Medical Publishers.
• Murray, R. K., Bender, D. A., Botham, K. M., Kennelly, P. J., Rodwell, V. W., & Weil, P. A. (2015). Harper's Illustrated Biochemistry (30th ed.). Lange Medical Books / McGraw-Hill Education.
• Nelson, D. L., & Cox, M. M. (2012). Lehninger Principles of Biochemistry (6th ed.). W. H. Freeman.
• Champe, P. C., & Harvey, R. A. (2013). Lippincott's Illustrated Reviews: Biochemistry (6th ed.). Lippincott Williams & Wilkins.
• Peet, A., Lieberman, M. A., & Marks, A. (2012). Marks' Basic Medical Biochemistry (4th ed.). Lippincott Williams & Wilkins.