Comprehensive Study Notes on Biomolecules: Structure, Function, and Enzymatic and Enzymatic Analysis

Comparison of Living and Non-Living Matter

• Living organisms possess a wide diversity of chemicals, including elements and compounds.

• Elemental Analysis of Living Tissue: Performing analysis on plant tissue, animal tissue, or microbial paste provides a list of elements such as carbon, hydrogen, oxygen, and others, along with their content per unit mass.

• Elemental Analysis of Non-Living Matter: Analysis of a piece of the earth’s crust yields a similar list of elements.

• Absolute vs. Relative Differences:

  • In absolute terms, all elements present in the earth’s crust are also present in living tissues.

  • In relative terms, a closer examination reveals that the relative abundance of carbon and hydrogen with respect to other elements is significantly higher in living organisms than in the earth's crust.

Elemental and Inorganic Composition Tables

• Table 9.1: Comparison of Elements in Earth’s Crust vs. Human Body (% Weight):

  • Hydrogen ($H$): Earth's Crust: $0.14$; Human Body: $9.5$

  • Carbon ($C$): Earth's Crust: $0.03$; Human Body: $18.5$

  • Oxygen ($O$): Earth's Crust: $46.6$; Human Body: $65.0$

  • Nitrogen ($N$): Earth's Crust: very little; Human Body: $3.3$

  • Sulphur ($S$): Earth's Crust: $0.03$; Human Body: $0.3$

  • Sodium ($Na$): Earth's Crust: $2.8$; Human Body: $0.2$

  • Calcium ($Ca$): Earth's Crust: $3.6$; Human Body: $1.5$

  • Magnesium ($Mg$): Earth's Crust: $2.1$; Human Body: $0.1$

  • Silicon ($Si$): Earth's Crust: $27.7$; Human Body: negligible

• Table 9.2: Inorganic Constituents of Living Tissues:

  • Sodium: $Na^{+}$

  • Potassium: $K^{+}$

  • Calcium: $Ca^{++}$

  • Magnesium: $Mg^{++}$

  • Water: $H_{2}O$

  • Compounds: $NaCl$, $CaCO_{3}$, $PO_{4}^{3-}$, $SO_{4}^{2-}$

Methods of Chemical Analysis for Organic Compounds

• To identify organic compounds, a living tissue (e.g., vegetable or liver) is ground in trichloroacetic acid ($Cl_{3}CCOOH$) using a mortar and pestle to create a thick slurry.

• The slurry is strained through cheesecloth or cotton, resulting in two fractions:

  • Filtrate (Acid-Soluble Pool): Contains thousands of organic compounds with molecular weights ranging from roughly $18$ to $800$ daltons ($Da$). It represents the cytoplasmic composition.

  • Retentate (Acid-Insoluble Fraction): Contains macromolecules like proteins, nucleic acids, and polysaccharides.

• Identification Procedure: Compounds are extracted, separated through various techniques, isolated, and purified. Analytical techniques reveal the molecular formula and probable structure.

• Biomolecules Definition: All carbon compounds obtained from living tissues are termed 'biomolecules'.

Analysis of Inorganic Compounds and Elements

• The Ash Method:

  1. Weigh a small amount of living tissue (wet weight).

  2. Dry the tissue to evaporate all water (dry weight).

  3. Fully burn the tissue to oxidize carbon compounds to gaseous form ($CO_{2}$, water vapour).

  4. The remaining material is named "ash."

• Content of Ash: Contains inorganic elements such as calcium and magnesium. Inorganic compounds like sulphate and phosphate are also found in the acid-soluble fraction.

Classification of Organic Biomolecules

• From a biological perspective, organic compounds are classified into amino acids, nucleotide bases, and fatty acids.

Amino Acids

• Structure: Organic compounds containing an amino group and an acidic (carboxyl) group on the same carbon, known as the $\alpha$-carbon. They are considered substituted methanes.

• Substituents: Four groups occupy the four valency positions of the $\alpha$-carbon: Hydrogen ($H$), Carboxyl group ($-COOH$), Amino group ($-NH_{2}$), and a variable group ($R$ group).

• Varieties: While many exist, only $20$ types occur in proteins.

  • Glycine: $R$ group is Hydrogen ($H$).

  • Alanine: $R$ group is a Methyl group ($-CH_{3}$).

  • Serine: $R$ group is Hydroxy methyl ($-CH_{2}OH$).

• Properties: Determined by the amino, carboxyl, and $R$ groups.

  • Acidic: e.g., Glutamic acid.

  • Basic: e.g., Lysine.

  • Neutral: e.g., Valine.

  • Aromatic: e.g., Tyrosine, Phenylalanine, Tryptophan.

• Zwitterionic Form: In solutions of different $pH$, the structure changes due to the ionizable nature of $-NH_{2}$ and $-COOH$ groups.

Lipids

• General Property: Water insoluble.

• Fatty Acids: Consist of a carboxyl group attached to an $R$ group.

  • The $R$ group can be methyl ($-CH_{3}$), ethyl ($-C_{2}H_{5}$), or a chain of $1$ to $19$ carbons.

  • Palmitic acid: $16$ carbons (including carboxyl carbon).

  • Arachidonic acid: $20$ carbons (including carboxyl carbon).

  • Saturated: No double bonds.

  • Unsaturated: One or more $C=C$ double bonds.

• Glycerol: Trihydroxy propane.

• Glycerides: Fatty acids esterified with glycerol.

  • Classified as monoglycerides, diglycerides, and triglycerides.

  • Called fats or oils based on melting point. Oils (e.g., gingelly oil) have lower melting points and remain liquid in winter.

• Phospholipids: Lipids containing phosphorus and a phosphorylated organic compound, found in cell membranes (e.g., Lecithin).

• Complex Lipids: Found in neural tissues with more intricate structures.

Nitrogen Bases, Nucleosides, and Nucleotides

• Nitrogen Bases: Heterocyclic rings including Adenine, Guanine, Cytosine, Uracil, and Thymine.

• Nucleosides: Nitrogen base attached to a sugar (e.g., Adenosine, Guanosine, Thymidine, Uridine, Cytidine).

• Nucleotides: Nucleoside with a phosphate group esterified to the sugar (e.g., Adenylic acid, Thymidylic acid, Guanylic acid, Uridylic acid, Cytidylic acid).

• Nucleic Acids: DNA and RNA consist of nucleotides and serve as genetic material.

Primary and Secondary Metabolites

• Primary Metabolites: Biomolecules involved in normal physiological processes with identifiable functions (e.g., amino acids, sugars).

• Secondary Metabolites: Found in plant, fungal, and microbial cells; functions are not always understood for the host but often have ecological importance or human utility.

• Table 9.3: Categories of Secondary Metabolites:

  • Pigments: Carotenoids, Anthocyanins.

  • Alkaloids: Morphine, Codeine.

  • Terpenoides: Monoterpenes, Diterpenes.

  • Essential oils: Lemon grass oil.

  • Toxins: Abrin, Ricin.

  • Lectins: Concanavalin A.

  • Drugs: Vinblastin, Curcumin.

  • Polymeric substances: Rubber, Gums, Cellulose.

Biomacromolecules

• Micromolecules: Found in the acid-soluble pool with molecular weight < 1000 daltons ($18$ to $800$ $Da$).

• Macromolecules/Biomacromolecules: Found in the acid-insoluble fraction with weights > 10,000 daltons.

  • Includes proteins, nucleic acids, and polysaccharides.

  • These are typically polymers.

• The Case of Lipids:

  • Molecular weights do not exceed $800$ $Da$.

  • They appear in the acid-insoluble fraction because they are part of membranes.

  • When tissue is ground, membranes break into water-insoluble vesicles that separate with the macromolecular fraction.

• Average Composition of Cells (Table 9.4):

  • Water: $70\text{-}90\%$

  • Proteins: $10\text{-}15\%$

  • Carbohydrates: $3\%$

  • Lipids: $2\%$

  • Nucleic acids: $5\text{-}7\%$

  • Ions: $1\%$

Proteins

• Definition: Polypeptides; linear chains of amino acids linked by peptide bonds.

• Heteropolymers: Proteins are polymers of $20$ different types of amino acids, not homopolymers.

• Nutritional Classification:

  • Essential Amino Acids: Cannot be made by the body; must be supplied through diet.

  • Non-essential Amino Acids: Can be synthesized by the body.

• Functions (Table 9.5):

  • Collagen: Intercellular ground substance; most abundant protein in the animal world.

  • Trypsin: Enzyme.

  • Insulin: Hormone.

  • Antibody: Fights infectious agents.

  • Receptor: Sensory reception (smell, taste).

  • GLUT-4: Enables glucose transport into cells.

  • RuBisCO: Ribulose bisphosphate Carboxylase-Oxygenase; most abundant protein in the whole biosphere.

Polysaccharides

• Structure: Long chains of sugars (monosaccharides) acting as building blocks.

• Homopolymers:

  • Cellulose: Polymer of glucose; found in plant cell walls, paper (plant pulp), and cotton fibre.

  • Starch: Storehouse of energy in plants; forms helical secondary structures and can hold $I_{2}$ molecules (forming a blue colour).

  • Glycogen: Storehouse of energy in animals.

  • Inulin: Polymer of fructose.

  • Chitin: Found in the exoskeletons of arthropods.

• Glycogen Structure: The right end is the reducing end, and the left end is the non-reducing end. It is highly branched.

• Complex Polysaccharides: May contain amino-sugars (e.g., glucosamine) or N-acetyl galactosamine.

Nucleic Acids

• Definition: Polynucleotides found in the acid-insoluble fraction.

• Components of a Nucleotide:

  1. Heterocyclic Compound: Nitrogenous bases (Purines: Adenine and Guanine; Pyrimidines: Uracil, Cytosine, Thymine).

  2. Monosaccharide: Ribose (in RNA) or 2' deoxyribose (in DNA).

  3. Phosphoric Acid/Phosphate: Esterified to the sugar.

Structure of Proteins

• Primary Structure: The linear sequence of amino acids. The first amino acid is the N-terminal and the last is the C-terminal.

• Secondary Structure: Folding of the chain into specific shapes like the right-handed $\alpha$-helix or $\beta$-pleated sheets.

• Tertiary Structure: The long protein chain folds upon itself like a hollow woolen ball, creating a 3D shape essential for biological activity.

• Quaternary Structure: The arrangement of multiple polypeptide subunits (e.g., Human Haemoglobin has 4 subunits: two $\alpha$ types and two $\beta$ types).

Enzymes: Biological Catalysts

• Nature: Almost all enzymes are proteins. Ribozymes are catalytic nucleic acids.

• Active Site: A crevice or pocket formed by the tertiary structure into which the substrate fits.

• Thermal Stability:

  • Inorganic catalysts work at high temperatures/pressures.

  • Most enzymes are damaged above $40^{\circ}C$.

  • Enzymes from thermophilic organisms (e.g., hot vents) are stable up to $80\text{-}90^{\circ}C$.

Chemical Reactions and Rates

• Rate: Amount of product ($P$) formed per unit time ($t$). $Rate = \frac{\delta P}{\delta t}$.

• Rule of Thumb: Rate doubles or halves for every $10^{\circ}C$ change in temperature.

• Catalytic Power Example:

  • $CO_{2} + H_{2}O \xrightarrow{\text{Carbonic anhydrase}} H_{2}CO_{3}$

  • Without enzyme: $200$ molecules/hour.

  • With enzyme: $600,000$ molecules/second ($10$ million times faster).

• Metabolic Pathway: A multistep reaction where each step is catalysed by enzymes (e.g., Glycolysis: Glucose to 2 Pyruvic acid via 10 steps).

Mechanism of Enzyme Action

• Substrate (S) to Product (P): The substrate binds at the active site forming a transient Enzyme-Substrate ($ES$) complex.

• Transition State: An unstable, high-energy intermediate state formed during bond breaking/making.

• Activation Energy: The difference between the average energy of the substrate and the energy of the transition state. Enzymes lower this energy barrier.

• Catalytic Cycle:

  1. Substrate binds to the active site.

  2. Binding induces a shape change in the enzyme (induced fit).

  3. Active site breaks/forms bonds to create the Enzyme-Product ($EP$) complex.

  4. Enzyme releases products and is free to bind again.

Factors Affecting Enzyme Activity

• Temperature and pH: Each enzyme has an optimum temperature and optimum $pH$. Higher temperatures denature proteins, while lower temperatures cause temporary inactivity.

• Substrate Concentration ($[S]$): Velocity increases until it reaches $V_{max}$, at which point all enzyme active sites are saturated.

• Inhibition:

  • Inhibitor: A chemical that shuts off activity.

  • Competitive Inhibitor: Closely resembles the substrate and competes for the active site (e.g., inhibition of succinic dehydrogenase by malonate, which resembles succinate).

Classification of Enzymes

1. Oxidoreductases/dehydrogenases: Catalyse S-reduced + S'-oxidised $\rightarrow$ S-oxidised + S'-reduced.

2. Transferases: Transfer a group ($G$) other than hydrogen: S-G + S' $\rightarrow$ S + S'-G.

3. Hydrolases: Catalyse hydrolysis of bonds (ester, ether, peptide, etc.).

4. Lyases: Remove groups without hydrolysis, leaving double bonds.

5. Isomerases: Inter-convert optical, geometric, or positional isomers.

6. Ligases: Link two compounds together (e.g., C-O, C-S, C-N, P-O bonds).

Enzyme Co-factors

• Apoenzyme: The protein portion of the enzyme.

• Co-factors: Non-protein constituents required for catalytic activity.

• Types of Co-factors:

  1. Prosthetic Groups: Tightly bound organic compounds (e.g., Haem in peroxidase and catalase).

  2. Co-enzymes: Transiently bound organic compounds, often containing vitamins (e.g., NAD and NADP contain niacin).

  3. Metal Ions: Form coordination bonds with the active site and substrate (e.g., Zinc for carboxypeptidase).