🧬 Biomolecules & Enzyme Kinetics

All living organisms are composed of organic macromolecules and inorganic constituents operating in a dynamic non-equilibrium steady state. Mastery of biomolecular thermodynamics and enzyme kinetics is essential for understanding physiological regulation and metabolic fluxes.

---

1. 🧪 Chemical Composition Analysis (Acid-Soluble vs Acid-Insoluble Pool)

When living tissue (e.g., liver or leaf) is ground in Trichloroacetic acid ($C{l}_{3}C-COOH$) and filtered through cheesecloth:

Fraction	State	Molecular Weight Range	Major Chemical Constituents
Filtrate (Acid-Soluble Pool)	Biomicromolecules / Cytoplasmic matrix	$18-800\text{Da}$	Monosaccharides, Amino acids, Nucleotides, Mineral ions, Organic acids
Retentate (Acid-Insoluble Pool)	Biomacromolecules / Cellular organelles	$>10,000\text{Da}$	Proteins, Polysaccharides, Nucleic Acids, Lipids

The Lipid Anomaly (High-Yield NEET Trap)

Lipids have molecular weights under $800\text{Da}$, yet they separate into the acid-insoluble retentate!

• Reason: Lipids are not polymeric macromolecules; however, when cell membranes are disrupted during grinding, lipids aggregate into insoluble hydrophobic vesicles and micelles that cannot pass through the filter mesh.

---

2. 🥩 Amino Acids & Protein Architecture

Amino acids are substituted methanes possessing an amino group ($-N{H}_2$), a carboxyl group ($-COOH$), hydrogen ($-H$), and a variable side chain ($-R$) on the $\alpha$-carbon.

       H
       |
H2N ── C ── COOH  <== Zwitterionic Form: H3N+ ── CH(R) ── COO-
       |
       R

2.1 Essential vs Non-Essential Amino Acids

• Essential Amino Acids (Must be acquired via diet):$$\text{PVT TIM HALL}⟹\text{Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine}$$
• Aromatic Amino Acids: Tyrosine, Phenylalanine, Tryptophan.
• Sulfur-Containing Amino Acids: Methionine, Cysteine.
• Basic Amino Acids: Lysine ($+1$), Arginine ($+1$), Histidine.
• Acidic Amino Acids: Glutamic acid ($-1$), Aspartic acid ($-1$).
• Neutral Amino Acids: Glycine (simplest, optically inactive), Alanine, Valine.

2.2 Hierarchical Levels of Protein Structure

Level	Stabilizing Chemical Bonds	Structural Motifs	Classic Examples
Primary ($1^{\circ }$)	Covalent peptide bonds ($-CO-NH-$)	Positional linear sequence from N-terminus to C-terminus	Insulin (51 amino acids), Polypeptide chain
Secondary ($2^{\circ }$)	Intramolecular & Intermolecular Hydrogen bonds	$\alpha$-Helix (Right-handed, $3.6\text{residues/turn}$), $\beta$-Pleated Sheets	Keratin (hair), Collagen, Fibroin (silk)
Tertiary ($3^{\circ }$)	Hydrophobic interactions, Disulfide bridges ($-S-S-$), Ionic bonds, Van der Waals	3D globular biological folding (Creates active catalytic clefts)	Myoglobin, Ribonuclease, Most enzymes
Quaternary ($4^{\circ }$)	Non-covalent subunit interactions	Multi-subunit spatial assembly	Hemoglobin (${\alpha }_2{\beta }_2$ tetramer, $4\text{heme groups}$)

---

3. ⚡ Enzyme Catalysis & Michaelis-Menten Kinetics

Enzymes are biocatalysts that accelerate chemical reactions by lowering the activation energy ($E_a$) without altering the overall thermodynamic equilibrium ($\mathrm{\Delta }{G}^{\circ }$).

$$\mathbf{E}\mathbf{+}\mathbf{S}\underset{{\mathbf{k}}_{\mathbf{-}\mathbf{1}}}{\overset{{\mathbf{k}}_{\mathbf{1}}}{\rightleftharpoons }}\mathbf{ES}\stackrel{{\mathbf{k}}_{\text{cat}}}{\to }\mathbf{E}\mathbf{+}\mathbf{P}$$

  Potential Energy (kJ/mol)
      ▲
      │       /───\  <-- Uncatalyzed Reaction (High Ea)
      │      /           │     /  /─\  \ <-- Enzyme-Catalyzed Reaction (Lower Ea)
      │    /  /   \        │   S  /     \        │  ───        \        │              \── P (Overall ΔG is IDENTICAL)
      └─────────────────────────────────► Reaction Coordinate

3.1 The Michaelis-Menten Equation

$$v=\frac{V_{max}[S]}{K_m+[S]}$$

Where:

• $v$ = Initial reaction velocity.
• $V_{max}$ = Maximal velocity when all active sites are saturated with substrate.
• $K_m$ (Michaelis Constant) = Substrate concentration $[S]$ at which the reaction velocity is half-maximal ($v=\frac{V_{max}}{2}$).
• Affinity Relationship:$${\mathbf{K}}_{\mathbf{m}}\propto \frac{\mathbf{1}}{\text{Enzyme Affinity for Substrate}}$$(A smaller $K_m$ signifies higher catalytic affinity).

---

4. 🛑 Enzyme Inhibition Mechanisms

                        ENZYME INHIBITION REGIMES
                                   │
         ┌─────────────────────────┴─────────────────────────┐
         ▼                                                   ▼
   [COMPETITIVE INHIBITION]                            [NON-COMPETITIVE INHIBITION]
   ├── Binds directly to catalytic Active Site         ├── Binds to Allosteric Regulatory Site
   ├── Structural analogue of substrate                ├── Changes catalytic conformation
   ├── Km INCREASES (Apparent affinity drops)          ├── Km UNCHANGED (Affinity untouched)
   └── Vmax UNCHANGED (Overcome by high [S])           └── Vmax DECREASES (Catalytic turnover halted)

Parameter	Competitive Inhibition	Non-Competitive Inhibition	Uncompetitive Inhibition
Binding Site	Catalytic active site	Allosteric site (E or ES)	ES complex exclusively
Substrate Overcome?	Yes (At high $[S]$, inhibitor displaced)	No (Cannot be reversed by adding $[S]$)	No
$V_{max}$ Effect	Unchanged	Decreased	Decreased
$K_m$ Effect	Increased	Unchanged	Decreased
Canonical Example	Malonate inhibiting Succinate Dehydrogenase; Statin inhibiting HMG-CoA reductase	Cyanide inhibiting Cytochrome c Oxidase ($F{e}^{3+}$ binding)	Lithium on inositol monophosphatase

---

5. 🧩 Enzyme Classification (IUBMB System: 6 Master Classes)

$$\text{Memory Mnemonic: }\text{OTHLIL}$$

1. Oxidoreductases / Dehydrogenases: Catalyze oxidation-reduction reactions ($A_{\text{red}}+B_{\text{ox}}\rightleftharpoons A_{\text{ox}}+B_{\text{red}}$).
2. Transferases: Transfer functional groups other than hydrogen ($S-G+S^{\prime }\rightleftharpoons S+S^{\prime }-G$).
3. Hydrolases: Cleave ester, ether, peptide, glycosidic, or $C-C$ bonds using water ($H_{2}O$).
4. Lyases: Cleave bonds by mechanisms other than hydrolysis, leaving double bonds ($C-C⟶C=C$).
5. Isomerases: Catalyze interconversion of geometric, optical, or structural isomers.
6. Ligases / Synthetases: Catalyze joining of two molecules coupled with ATP hydrolysis (e.g., DNA Ligase, Glutamine synthetase).