Biomolecules

How to Analyse Chemical Composition?

Living organisms are composed of various chemical substances. To understand the chemistry of life, it is essential to analyse the chemical composition of living tissues.

Chemical Analysis of Living Tissue

A standard procedure to analyse the chemical composition of a living tissue (like a piece of liver, a piece of vegetable) is as follows:

1. Take a small piece of living tissue (e.g., 10-20 grams).

2. Grind it in a mortar and pestle using trichloroacetic acid ($Cl_3CCOOH$). This process homogenizes the tissue.

3. A thick slurry is obtained. Strain this slurry through a piece of cheesecloth or cotton.

4. Two fractions are obtained:

• The fluid that passes through is called the filtrate or the acid-soluble pool. This fraction contains relatively smaller biomolecules (micromolecules).
• The material retained on the cheesecloth/cotton is called the retentate or the acid-insoluble fraction. This fraction contains larger biomolecules (macromolecules).

*(Image shows the steps: grinding tissue in trichloroacetic acid, straining, separating acid-soluble filtrate and acid-insoluble retentate)*

Components of the Fractions

Acid-soluble pool (Micromolecules):

• These are molecules with molecular weights generally ranging from 18 to around 800 Daltons (Da).
• They include:
  • Simple sugars (monosaccharides) and disaccharides.
  • Amino acids.
  • Nitrogen bases (purines and pyrimidines).
  • Nucleosides and nucleotides.
  • Fatty acids.
  • Glycerol.
  • Some vitamins.
  • Mineral ions.
  • Water.

Acid-insoluble fraction (Macromolecules):

• These are polymeric substances with high molecular weights, typically 10,000 Daltons and above.
• They include:
  • Proteins (polymers of amino acids).
  • Polysaccharides (polymers of monosaccharides).
  • Nucleic acids (DNA and RNA - polymers of nucleotides).
  • Lipids are also found in the acid-insoluble fraction, but they are not true macromolecules or polymers. They are compounds with molecular weights generally not exceeding 800 Da. Lipids are found in the acid-insoluble fraction because they form vesicles (membrane fragments) when the tissue is ground, and these vesicles are insoluble in trichloroacetic acid.

Elemental Analysis

Elemental analysis gives a list of elements present in living tissue, like Carbon (C), Hydrogen (H), Oxygen (O), Nitrogen (N), Sulphur (S), Phosphorus (P), etc. This is done by burning a piece of living tissue.

• When a tissue is burnt, all the carbon compounds are oxidised to gaseous form ($CO_2$) and removed.
• The water ($H_2O$) evaporates.
• The remaining inorganic matter forms the ash. The ash contains inorganic elements present as inorganic compounds (e.g., Calcium phosphate, Sodium chloride, Potassium sulphate).

Comparison of elemental composition of living tissue and Earth's crust (non-living matter):

*(Source: NCERT Biology Class 11)*

This comparison shows that Carbon and Hydrogen are found in much higher amounts in living organisms than in the Earth's crust, which is expected since biomolecules are carbon-based organic compounds.

Inorganic Constituents

While the acid-soluble pool contains organic micromolecules, it also contains inorganic compounds. These include:

• Mineral ions (e.g., $Na^+, K^+, Ca^{2+}, Mg^{2+}, Cl^-, PO_4^{3-}, SO_4^{2-}$).
• Gases (e.g., $O_2, CO_2$).
• Water.

These inorganic substances are also essential components of living matter and are found in the acid-soluble fraction when analysed using the trichloroacetic acid method (except for gases and water which are removed differently).

Primary and Secondary Metabolites

All the chemicals found in living organisms are called biomolecules. These biomolecules are found in the acid-soluble pool and acid-insoluble fraction as discussed in the previous section.

Primary Metabolites

These are biomolecules that have identifiable, known functions and play direct roles in normal growth, development, and reproduction of organisms.

• They are essential for the survival of the organism.
• Examples include amino acids, nucleotides, sugars, fatty acids, glycerol, vitamins, etc.
• These are typically found in the acid-soluble pool (micromolecules). Macromolecules like proteins, nucleic acids, and polysaccharides, which are polymers of primary metabolites, are found in the acid-insoluble fraction.

Secondary Metabolites

Many organisms, especially microbes, fungi, and plants, produce biomolecules that are not directly involved in their primary metabolic processes like growth, development, or reproduction. These are called secondary metabolites.

• Their role in the producer organism is not always fully understood, but they are often thought to be involved in defence, communication, or adaptation to the environment.
• From a human perspective, many secondary metabolites have significant economic importance (e.g., drugs, pigments, spices, perfumes, insecticides).
• Examples are numerous and diverse.

Examples of Secondary Metabolites

*(Note: Some substances like rubber, gums, cellulose could be considered both structural/primary in plants, but are often listed as secondary when discussing diverse compounds from different classes for various purposes)*

The acid-soluble pool primarily consists of primary metabolites and inorganic constituents. The acid-insoluble fraction primarily consists of macromolecules (polymers of primary metabolites) and lipids (which are small molecules but precipitate).

Biomacromolecules

As seen from the chemical analysis of living tissues, the acid-insoluble fraction contains molecules with high molecular weights. These large molecules are called biomacromolecules.

Biomacromolecules are typically formed by the polymerization of smaller repeating units (monomers).

The major classes of biomacromolecules found in the acid-insoluble fraction are:

1. Proteins: Polymers of amino acids.
2. Polysaccharides: Polymers of monosaccharides (simple sugars).
3. Nucleic Acids (DNA and RNA): Polymers of nucleotides.

Lipids are also found in the acid-insoluble fraction, but they are not polymers in the strict sense. They are formed by the esterification of fatty acids and glycerol (or other alcohols). Their molecular weight is generally much lower than proteins, polysaccharides, or nucleic acids, yet they are included in the acid-insoluble fraction because of their insolubility in the acidic solvent used for extraction.

Characteristics of Biomacromolecules

• They are large molecules with molecular weights typically in the range of thousands to millions of Daltons.
• They are formed by linking smaller monomer units through specific covalent bonds.
• They are generally found in the acid-insoluble fraction during tissue analysis.
• They perform crucial functions in the cell, including structure, catalysis, storage, information transfer, etc.

Answer:

Proteins

Proteins are biomacromolecules that are the most abundant organic molecules in living organisms. They are essential for almost every function within a cell.

Monomers of Proteins: Amino Acids

Proteins are linear chains of amino acids linked together by peptide bonds.

Amino acids are organic compounds that contain both an amino group $(-\text{NH}_2)$ and a carboxyl group $(-\text{COOH})$ attached to the same carbon atom, called the alpha ($\alpha$) carbon. They also have a hydrogen atom and a variable side chain ($R$ group) attached to the $\alpha$ carbon.

General structure of an amino acid:

$ \begin{smallmatrix} \text{H} \\ | \\ \text{H}_2\text{N} - \underset{\alpha}{\text{C}} - \text{COOH} \\ | \\ \text{R} \end{smallmatrix} $

The $R$ group is what distinguishes one amino acid from another. There are 20 different types of amino acids commonly found in proteins. The $R$ group can be a simple hydrogen atom (Glycine), a methyl group (Alanine), or more complex structures (e.g., Serine, Cysteine, Tyrosine, etc.).

Classification of Amino Acids (based on R group)

Amino acids can be classified based on the chemical nature of their $R$ groups:

• Aliphatic: Glycine, Alanine, Valine, Leucine, Isoleucine.
• Aromatic: Phenylalanine, Tyrosine, Tryptophan.
• Acidic: Aspartic acid, Glutamic acid.
• Basic: Lysine, Arginine, Histidine.
• Hydroxyl-containing: Serine, Threonine.
• Sulphur-containing: Cysteine, Methionine.
• Imino acid: Proline (has an imino group instead of an amino group).

Amphoteric Nature of Amino Acids

Amino acids are amphoteric, meaning they can act as both acids (due to -COOH group) and bases (due to -$NH_2$ group).

In aqueous solution, at physiological pH, the carboxyl group is deprotonated $(-\text{COO}^-)$ and the amino group is protonated $(-\text{NH}_3^+)$. This form, with both positive and negative charges, is called a zwitterion.

$ \begin{smallmatrix} \text{H} \\ | \\ ^+\text{H}_3\text{N} - \underset{\alpha}{\text{C}} - \text{COO}^- \\ | \\ \text{R} \end{smallmatrix} $ (Zwitterion form)

The pH at which an amino acid exists as a zwitterion with no net charge is called its isoelectric point (pI).

The Peptide Bond

Amino acids are linked together by a peptide bond to form peptides and proteins.

A peptide bond is formed between the carboxyl group of one amino acid and the amino group of the next amino acid with the elimination of a water molecule (dehydration synthesis or condensation reaction).

$ \text{Amino acid}_1 - \text{COOH} + \text{NH}_2 - \text{Amino acid}_2 \rightarrow \text{Amino acid}_1 - \text{CO} - \text{NH} - \text{Amino acid}_2 + H_2O $

The $-\text{CO}-\text{NH}-$ linkage is the peptide bond.

• Two amino acids linked by a peptide bond form a dipeptide.
• A chain of several amino acids is called a polypeptide.
• Proteins are long polypeptide chains, often containing hundreds or thousands of amino acids, that fold into specific three-dimensional structures.

*(Image shows two amino acids undergoing condensation reaction to form a dipeptide with a peptide bond and release water)*

Functions of Proteins

Proteins are incredibly versatile molecules, performing a wide range of functions in living organisms:

• Enzymes: Most enzymes, which catalyze biochemical reactions, are proteins (e.g., Trypsin).
• Structural components: Provide support and shape (e.g., Collagen is the most abundant protein in the animal world, Keratin in hair and nails).
• Transport: Carry substances (e.g., Haemoglobin transports oxygen in blood, Carrier proteins transport molecules across cell membranes).
• Movement: Essential for muscle contraction (e.g., Actin, Myosin).
• Immune defence: Antibodies (immunoglobulins) are proteins that protect against pathogens.
• Hormones: Some hormones are proteins (e.g., Insulin, Glucagon).
• Receptors: Proteins on cell surfaces that bind to specific molecules for communication.

Polysaccharides

Polysaccharides are complex carbohydrates that are long chains (polymers) of repeating monosaccharide (simple sugar) units linked together by glycosidic bonds.

Monomers of Polysaccharides: Monosaccharides

Monosaccharides are the simplest sugars. They are classified based on the number of carbon atoms (e.g., trioses (3C), tetroses (4C), pentoses (5C), hexoses (6C)) and the type of carbonyl group (aldehyde in aldoses, ketone in ketoses).

Examples: Glucose, Fructose, Galactose (hexoses); Ribose, Deoxyribose (pentoses).

The Glycosidic Bond

Monosaccharides are linked together by a glycosidic bond. This bond is formed between the hydroxyl groups of two monosaccharide units with the elimination of a water molecule (dehydration synthesis).

Example: Formation of Maltose (a disaccharide) from two glucose units:

$ \text{Glucose} - \text{OH} + \text{HO} - \text{Glucose} \rightarrow \text{Glucose} - \text{O} - \text{Glucose} + H_2O $

The oxygen bridge $(-\text{O}-)$ connecting the sugar units is the glycosidic bond. In polysaccharides, these bonds extend to form long chains.

*(Image shows two glucose molecules reacting to form maltose and water, highlighting the glycosidic bond)*

Classification of Polysaccharides

Polysaccharides can be classified based on their composition and structure:

1. Homopolysaccharides: Composed of only one type of monosaccharide monomer.
   • Starch: Storage polysaccharide in plants. Composed of glucose units. It is a mixture of Amylose (unbranched, $\alpha-1,4$ glycosidic bonds) and Amylopectin (branched, $\alpha-1,4$ and $\alpha-1,6$ glycosidic bonds).
   • Glycogen: Storage polysaccharide in animals (mainly in liver and muscles) and fungi. Composed of glucose units. It is highly branched with $\alpha-1,4$ and $\alpha-1,6$ glycosidic bonds, similar to amylopectin but more highly branched.
   • Cellulose: Structural polysaccharide in plant cell walls. Composed of glucose units linked by $\beta-1,4$ glycosidic bonds. This linkage makes it difficult to digest for most animals (except ruminants and termites with symbiotic microbes). It is a linear, unbranched polymer.
   • Inulin: A homopolysaccharide of fructose. Used as a storage carbohydrate in some plants (e.g., dahlia tubers, chicory). It is a linear polymer. It is used to estimate glomerular filtration rate (GFR) as it is freely filtered by kidneys and not reabsorbed.
   • Chitin: Structural polysaccharide found in the exoskeleton of arthropods and cell walls of fungi. Composed of N-acetylglucosamine (a modified glucose derivative) units. It is a linear, unbranched polymer.
2. Heteropolysaccharides: Composed of two or more different types of monosaccharides or modified monosaccharide units.
   • Examples: Peptidoglycan (in bacterial cell walls - contains sugars and amino acids), Hyaluronic acid (in connective tissues), Chondroitin sulphate (in cartilage).

Functions of Polysaccharides

• Energy Storage: Starch (plants), Glycogen (animals, fungi).
• Structural Support: Cellulose (plant cell walls), Chitin (fungal cell walls, arthropod exoskeleton), Peptidoglycan (bacterial cell walls).
• Lubrication and cushioning (e.g., Hyaluronic acid in joints).

Nucleic Acids

Nucleic acids are biomacromolecules that carry and transmit genetic information. The two main types of nucleic acids are Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA).

Monomers of Nucleic Acids: Nucleotides

Nucleic acids are polymers of nucleotides linked together by phosphodiester bonds.

Each nucleotide is composed of three parts:

1. A Nitrogenous Base:
   • Purines: Adenine (A), Guanine (G).
   • Pyrimidines: Cytosine (C), Thymine (T) in DNA, Uracil (U) in RNA.
2. A Pentose Sugar:
   • Deoxyribose in DNA.
   • Ribose in RNA. (Ribose has a hydroxyl (-OH) group at the 2' carbon, while deoxyribose has just a hydrogen (-H) at that position).
3. A Phosphate Group: $(PO_4^{3-})$.

Nucleosides and Nucleotides

• Nucleoside: A nitrogenous base linked to a pentose sugar. (Base + Sugar).
  • Examples: Adenosine (Adenine + Ribose), Guanosine (Guanine + Ribose), Cytidine (Cytosine + Ribose), Uridine (Uracil + Ribose) - these are ribonucleosides.
  • Deoxyadenosine (Adenine + Deoxyribose), Deoxyguanosine (Guanine + Deoxyribose), Deoxycytidine (Cytosine + Deoxyribose), Deoxythymidine (Thymine + Deoxyribose) - these are deoxyribonucleosides.
• Nucleotide: A nucleoside with one or more phosphate groups attached, usually to the 5' carbon of the sugar. (Base + Sugar + Phosphate).
  • Examples: Adenylic acid (Adenosine monophosphate - AMP), Guanylic acid (GMP), Cytidylic acid (CMP), Uridylic acid (UMP) - these are ribonucleotides.
  • Deoxyadenylic acid (dAMP), Deoxyguanylic acid (dGMP), Deoxycytidylic acid (dCMP), Deoxythymidylic acid (dTMP) - these are deoxyribonucleotides.

Nucleotides are the monomer units that are linked together to form nucleic acids.

*(Image shows a general nucleotide structure highlighting the base, pentose sugar, and phosphate group)*

The Phosphodiester Bond

Nucleotides are linked together in a nucleic acid chain by a phosphodiester bond.

This bond is formed between the phosphate group of one nucleotide and the hydroxyl group on the 3' carbon of the sugar of the next nucleotide, with the elimination of water.

The sugar-phosphate backbone of a nucleic acid strand is formed by these phosphodiester linkages. A nucleic acid strand has a directionality, with a 5' end (free phosphate group) and a 3' end (free hydroxyl group on the sugar).

*(Image shows two nucleotides linking via a phosphodiester bond, forming a dinucleotide and releasing water)*

DNA vs. RNA

DNA typically exists as a double helix (Watson-Crick model), where two antiparallel strands are held together by hydrogen bonds between complementary bases (A pairs with T with 2 hydrogen bonds; G pairs with C with 3 hydrogen bonds).

*(Image shows the DNA double helix with base pairing A-T and G-C and the two antiparallel sugar-phosphate backbones)*

Structure of Proteins

Proteins are not just simple linear chains of amino acids. They fold into specific three-dimensional structures that are essential for their function. The structure of a protein can be described at four levels of organisation.

Primary Structure

The primary structure is the linear sequence of amino acids in a polypeptide chain. This sequence is determined by the genetic code (DNA sequence). The amino acids are linked by peptide bonds.

Example: Ala-Gly-Ser-Val-...

The primary structure is the foundation for the higher levels of protein structure and ultimately determines the protein's function. A change in even a single amino acid in the primary sequence can sometimes drastically alter the protein's structure and function (e.g., sickle cell anaemia is caused by a single amino acid substitution in haemoglobin).

Secondary Structure

The secondary structure refers to the local folding of the polypeptide chain into specific shapes, primarily due to hydrogen bonds forming between the amino group of one amino acid and the carboxyl group of another further down the chain.

The most common types of secondary structures are:

• Alpha Helix ($\alpha$-helix): A coiled structure where the polypeptide chain forms a helix. Hydrogen bonds form between the peptide bond oxygen of one amino acid and the hydrogen atom of the peptide bond nitrogen of an amino acid four residues away.
• Beta-Pleated Sheet ($\beta$-sheet): A folded, sheet-like structure formed by segments of polypeptide chains lying side-by-side (either parallel or antiparallel) and connected by hydrogen bonds between peptide bonds.

*(Image shows representations of an alpha helix and a beta-pleated sheet)*

Tertiary Structure

The tertiary structure is the overall three-dimensional shape of a single polypeptide chain. This shape results from the folding and coiling of the secondary structure, driven and stabilized by interactions between the side chains ($R$ groups) of the amino acids.

Types of interactions stabilising tertiary structure:

• Disulfide bonds: Covalent bonds formed between the sulphur atoms of two cysteine residues.
• Ionic bonds (Salt bridges): Electrostatic attraction between positively charged basic amino acid side chains and negatively charged acidic amino acid side chains.
• Hydrogen bonds: Between polar side chains.
• Hydrophobic interactions: Nonpolar side chains tend to cluster together in the interior of the protein, away from water.
• Van der Waals forces: Weak interactions between atoms in close proximity.

The tertiary structure is crucial for the protein's function, especially for enzymes where the active site's shape is determined by the precise 3D arrangement of amino acid residues.

*(Image shows a folded polypeptide chain illustrating interactions like disulfide bonds, ionic bonds, hydrogen bonds, and hydrophobic interactions)*

Quaternary Structure

The quaternary structure exists only in proteins that are made up of more than one polypeptide subunit. It is the arrangement of these multiple polypeptide subunits in a functional protein complex.

The subunits are held together by various non-covalent interactions (hydrogen bonds, ionic bonds, hydrophobic interactions, Van der Waals forces) and sometimes disulfide bonds.

Example: Haemoglobin is a protein with quaternary structure. It consists of four polypeptide subunits: two alpha chains and two beta chains.

*(Image shows simplified representations of primary, secondary (helix, sheet), tertiary (folded chain), and quaternary (multiple folded chains associating) structures)*

Denaturation

The specific 3D structure of a protein can be disrupted by factors like heat, extreme pH, or certain chemicals. This process is called denaturation. Denaturation typically affects the secondary, tertiary, and quaternary structures, while the primary structure (amino acid sequence) usually remains intact.

Denaturation often leads to the loss of the protein's biological function (e.g., cooking an egg denatures the egg white protein albumin).

Nature of Bond linking Monomers in a Polymer

Biomacromolecules (except lipids) are polymers formed by joining many small monomer units. The monomers are linked together by specific covalent bonds formed through dehydration synthesis (removal of a water molecule).

Glycosidic Bond (in Polysaccharides)

Formed between two monosaccharide units. It is a covalent bond formed between the hydroxyl groups of two adjacent sugar molecules with the elimination of water. The bond is formed between the carbon atoms of the sugar rings, typically between C1 of one sugar and a hydroxyl group on another carbon (e.g., C4 or C6) of the adjacent sugar.

$ \text{Monosaccharide}_1 - \text{OH} + \text{HO} - \text{Monosaccharide}_2 \rightarrow \text{Monosaccharide}_1 - \text{O} - \text{Monosaccharide}_2 + H_2O $

Example: Bonds in Starch, Glycogen, Cellulose, Chitin.

Peptide Bond (in Proteins)

Formed between two amino acid units. It is a covalent bond formed between the carboxyl group $(-\text{COOH})$ of one amino acid and the amino group $(-\text{NH}_2)$ of the next amino acid with the elimination of water.

$ \text{Amino acid}_1 - \text{COOH} + \text{NH}_2 - \text{Amino acid}_2 \rightarrow \text{Amino acid}_1 - \text{CO} - \text{NH} - \text{Amino acid}_2 + H_2O $

Example: The bonds linking amino acids in polypeptide chains.

Phosphodiester Bond (in Nucleic Acids)

Formed between two nucleotide units. It is a covalent bond linking the 5' carbon of the sugar of one nucleotide to the 3' carbon of the sugar of the next nucleotide through a phosphate group.

Specifically, it is an ester bond formed between the phosphate group (attached to the 5' carbon of one sugar) and the hydroxyl group at the 3' carbon of the sugar of the next nucleotide.

$ \text{Nucleotide}_1 \:(5'-\text{Phosphate}) + \text{Nucleotide}_2 \:(3'-\text{OH}) \rightarrow \text{Nucleotide}_1 - \text{Phosphate} - \text{Nucleotide}_2 + H_2O $

Example: The backbone linkage in DNA and RNA strands.

Ester Bond (in Lipids)

While not polymers in the strict sense, lipids like triglycerides are formed by the esterification of fatty acids and glycerol. An ester bond is formed between the carboxyl group of a fatty acid and the hydroxyl group of glycerol (an alcohol) with the elimination of water.

$ \text{Fatty acid} - \text{COOH} + \text{HO} - \text{Alcohol (e.g., Glycerol)} \rightarrow \text{Fatty acid} - \text{CO} - \text{O} - \text{Alcohol} + H_2O $

Example: Bonds linking fatty acids to glycerol in fats and oils.

These specific covalent bonds are formed during the synthesis of biomacromolecules (anabolic processes) and are broken during their breakdown (catabolic processes) through hydrolysis (addition of water).

Dynamic State of Body Constituents - Concept of Metabolism

Living organisms are not static. They are constantly changing systems. The biomolecules that make up a living organism are continuously being synthesized and broken down. This constant turnover of biomolecules is known as the dynamic state of body constituents.

Metabolism: The Sum Total of Chemical Reactions

All the chemical reactions that occur within a living organism are collectively called metabolism.

• These reactions transform biomolecules from one form to another.
• They are often linked in sequences called metabolic pathways.
• Metabolic pathways can be linear, branched, or circular.

Types of Metabolic Reactions

Metabolism consists of two main types of processes:

• Anabolism (Biosynthesis):
  • These are constructive or synthetic reactions.
  • They involve the formation of complex substances from simpler ones.
  • Anabolic reactions consume energy (endergonic).
  • Examples: Synthesis of proteins from amino acids, synthesis of polysaccharides from monosaccharides, synthesis of DNA from nucleotides.
• Catabolism (Breakdown):
  • These are destructive or breakdown reactions.
  • They involve the breakdown of complex substances into simpler ones.
  • Catabolic reactions release energy (exergonic). This energy is often stored in the form of ATP.
  • Examples: Breakdown of glucose to lactic acid (glycolysis), breakdown of proteins into amino acids, breakdown of fats into fatty acids and glycerol.

*(Image shows a simple diagram with complex molecule -> simple molecules (catabolism, releasing energy) and simple molecules -> complex molecule (anabolism, consuming energy))*

The dynamic state means that even while an organism appears stable, its internal composition is constantly changing due to simultaneous anabolic and catabolic reactions occurring at all times.

For example, glucose is constantly being broken down to produce energy (catabolism), while new glucose molecules might be synthesized from other precursors (anabolism), or incorporated into glycogen for storage (anabolism).

Metabolic Basis for Living

Metabolism is the fundamental basis of life. It is the sum total of chemical reactions that sustain the living state.

Energy Transformations

One of the key aspects of metabolism is the transformation of energy. Catabolic pathways release energy stored in chemical bonds of food molecules. This energy is then captured and stored in a usable form, primarily as ATP (Adenosine Triphosphate).

ATP is the main energy currency of the cell. Anabolic pathways and other energy-requiring processes (like muscle contraction, nerve impulse transmission, active transport) utilise the energy released from the hydrolysis of ATP.

$ \text{ATP} \rightarrow \text{ADP} + \text{Pi} + \text{Energy} $

where ADP is Adenosine Diphosphate and Pi is inorganic phosphate.

Building and Recycling

Metabolism also involves the constant synthesis (anabolism) of new cellular components and the breakdown (catabolism) of old or damaged ones. This ensures that the cell is continuously renewed and can adapt to changing conditions.

Think of a factory where raw materials are brought in, processed into products, and waste is removed. Similarly, the cell takes in nutrients, metabolizes them to produce energy and building blocks, synthesizes new structures, and removes waste.

Metabolic Pathways and Regulations

Metabolic reactions are organised into intricate pathways, where the product of one reaction becomes the substrate for the next. These pathways are highly regulated to maintain homeostasis (a stable internal environment).

The regulation is primarily achieved through the control of enzymes, which are the catalysts for most metabolic reactions.

The flow of molecules through metabolic pathways is not random; it is directed and controlled to meet the cell's specific needs at any given time.

Answer:

The Living State

What constitutes the 'living state'? It is a complex interplay of various processes that maintain an organism as a distinct entity, constantly interacting with its environment.

The Dynamic State and Steady State

As discussed, living organisms are in a dynamic state due to continuous metabolism. Biomolecules are constantly changing. However, while individual molecules are being made and broken down, the overall concentration of most biomolecules within the cell or organism remains relatively constant over time in a healthy state. This is known as a steady state.

A steady state is a non-equilibrium state where the rate of influx or synthesis of a substance equals the rate of efflux or breakdown. It requires continuous work and energy input (from metabolism) to maintain.

Non-equilibrium Steady State

Living systems are open systems that constantly exchange matter and energy with their surroundings. They maintain a steady state that is far from equilibrium.

If a living system reached equilibrium, metabolism would cease, and the organism would die. Equilibrium is a state where there is no net change and no energy flow, which is incompatible with life.

Metabolic reactions and pathways are geared towards creating and maintaining this non-equilibrium steady state by constantly driving reactions in one direction, preventing them from reaching equilibrium. This requires a continuous supply of energy.

Living Process as a Non-equilibrium Steady State System

The living state is a self-organising, non-equilibrium steady state system capable of metabolism, response to stimuli, growth, adaptation, and reproduction. It requires a constant input of energy to maintain its organization and distance from equilibrium.

The energy required to prevent a living system from falling into equilibrium is obtained through metabolism, primarily through catabolic processes that release energy from food molecules.

In summary, the living state is characterised by:

• A dynamic state of biomolecules (continuous turnover).
• A steady state of component concentrations (rates of synthesis = rates of degradation).
• Operating as a non-equilibrium system, requiring constant energy input to maintain order and function.

Without metabolism, the dynamic state and the non-equilibrium steady state cannot be maintained, leading to a state of equilibrium (death).

Enzymes

Enzymes are biological catalysts that are essential for almost all biochemical reactions occurring in living organisms. They are responsible for the incredibly high rates and specificity of metabolic reactions.

Chemical Reactions

A chemical reaction involves the transformation of a substance (or substances) into another substance (or substances).

Example:

$ \text{Reactant(s)} \rightarrow \text{Product(s)} $

Chemical reactions can be either inorganic or organic. Reactions occurring in living systems are primarily organic chemical reactions.

Rate of Reactions

The rate of a chemical reaction is the amount of product formed per unit time. This rate can be influenced by factors like temperature, pressure, and the presence of catalysts.

$ \text{Rate} = \frac{\Delta \text{Product}}{\Delta \text{Time}} $ or $ \frac{-\Delta \text{Reactant}}{\Delta \text{Time}} $

Activation Energy

For a chemical reaction to occur, the reactant molecules must overcome an energy barrier. This energy barrier is called the activation energy ($E_a$). It is the minimum amount of energy required to start a chemical reaction or to convert a reactant into a transition state.

Transition state is an unstable, high-energy state that reactants must pass through before converting to products.

*(Image shows an energy profile diagram with Free Energy on Y-axis and Reaction Progress on X-axis, showing reactants, transition state (peak), products, and indicating activation energy as the energy difference between reactants and the transition state)*

How do Enzymes bring about such High Rates of Chemical Conversions?

Enzymes are catalysts, meaning they increase the rate of a chemical reaction without being consumed in the process.

Enzymes achieve this by lowering the activation energy required for the reaction to proceed. They do not change the equilibrium of the reaction, only how fast it reaches equilibrium.

*(Image shows an energy profile diagram comparing the activation energy barrier of an uncatalyzed reaction (higher peak) and an enzyme-catalyzed reaction (lower peak). Products and reactants are at the same energy levels in both cases)*

Enzymes are typically proteins (though some RNA molecules called ribozymes also have catalytic activity).

They are highly specific in their action, usually catalysing only one or a very limited number of reactions.

Nature of Enzyme Action

The mechanism of enzyme action involves the binding of the enzyme to its substrate(s) at a specific region called the active site.

The active site is a three-dimensional crevice or pocket formed by the folding of the polypeptide chain(s). It contains specific amino acid residues that are involved in binding the substrate and catalysing the reaction.

Steps in Enzyme Action

1. Binding of Substrate: The substrate (S) binds to the active site of the enzyme (E) to form an enzyme-substrate complex (ES). This binding is often compared to a "lock and key" mechanism or an "induced fit" model, where the active site changes shape slightly to accommodate the substrate.

$ E + S \rightleftharpoons ES $

2. Formation of Enzyme-Product Complex: Within the ES complex, the enzyme facilitates the chemical transformation of the substrate(s) into product(s) (P). The transition state is formed here, and the enzyme lowers its energy.

$ ES \rightarrow EP $

3. Release of Product: The product(s) dissociate from the enzyme's active site, leaving the enzyme free to bind another substrate molecule.

$ EP \rightarrow E + P $

The overall reaction catalysed by an enzyme can be summarised as:

$ E + S \rightleftharpoons ES \rightarrow EP \rightarrow E + P $

*(Image shows sequential steps: free enzyme and substrate, substrate binding to active site, transition state/EP complex, release of product and free enzyme)*

Factors Affecting Enzyme Activity

The activity of an enzyme, i.e., its rate of catalysis, can be affected by several factors:

Temperature

• Enzymes work within a specific range of temperatures.
• Enzyme activity generally increases with temperature up to an optimum temperature. This is because higher temperature increases the kinetic energy of molecules, leading to more frequent collisions between enzyme and substrate.
• Beyond the optimum temperature, enzyme activity decreases sharply. High temperatures cause the protein structure of the enzyme to denature, losing its specific 3D shape and thus its active site and catalytic ability.
• Each enzyme has an optimum temperature where its activity is maximal. For human enzymes, this is typically around $37^\circ C$.

*(Image shows a bell-shaped curve with enzyme activity on the Y-axis and temperature on the X-axis, peaking at optimum temperature)*

pH

• Enzymes are also sensitive to pH changes.
• Enzyme activity is maximal at an optimum pH.
• Changing the pH away from the optimum can affect the ionization state of amino acid residues in the active site and the substrate, altering their ability to interact and thus reducing enzyme activity.
• Extreme pH values can lead to denaturation of the enzyme protein.
• Different enzymes have different optimum pH values (e.g., Pepsin in the stomach works best at acidic pH $\approx 1.5-2.5$, Trypsin in the small intestine works best at alkaline pH $\approx 8.0$).

*(Image shows a bell-shaped curve with enzyme activity on the Y-axis and pH on the X-axis, peaking at optimum pH)*

Concentration of Substrate

• At low substrate concentrations, the reaction rate increases as substrate concentration increases. This is because more substrate molecules are available to bind to enzyme active sites.
• As the substrate concentration is increased, the reaction rate eventually reaches a maximum velocity ($V_{max}$). At this point, all the enzyme active sites are saturated with substrate molecules, and the enzyme is working at its maximum capacity.
• Increasing substrate concentration further beyond $V_{max}$ will not increase the rate, unless more enzyme is added.

*(Image shows a hyperbolic curve with reaction rate (or initial velocity $V_0$) on the Y-axis and substrate concentration [S] on the X-axis, showing the rate increasing then levelling off at $V_{max}$)*

Inhibitors

The activity of an enzyme can be decreased or stopped by certain chemicals called inhibitors.

• Competitive inhibitors: Molecules that are structurally similar to the substrate and compete for binding to the active site of the enzyme. They can be overcome by increasing the substrate concentration.
• Non-competitive inhibitors: Molecules that bind to a different site on the enzyme (not the active site) called the allosteric site, causing a conformational change in the enzyme that reduces its catalytic activity. Increasing substrate concentration does not overcome this inhibition.

Classification and Nomenclature of Enzymes

Enzymes are named and classified based on the type of reaction they catalyse. A widely accepted system is the classification given by the International Union of Biochemistry (IUB).

IUB Classification System

Enzymes are divided into 6 classes, each with several subclasses and sub-subclasses. Each enzyme is assigned a unique 4-digit EC number (Enzyme Commission number).

The 6 main classes are:

1. Oxidoreductases/Dehydrogenases: Catalyse redox reactions (transfer of electrons or hydrogen atoms).

   Example: Alcohol dehydrogenase.

2. Transferases: Catalyse the transfer of a functional group (other than hydrogen) from one molecule to another.

   Example: Hexokinase (transfers a phosphate group from ATP to glucose).

3. Hydrolases: Catalyse hydrolysis reactions (breaking of bonds using water).

   Example: Amylase, Lipase, Protease.

4. Lyases: Catalyse the removal of groups from substrates by mechanisms other than hydrolysis, often resulting in the formation of double bonds.

   Example: Aldolase, Adenylate cyclase.

5. Isomerases: Catalyse the rearrangement of atoms within a molecule to form isomers.

   Example: Glucose-6-phosphate isomerase.

6. Ligases: Catalyse the joining of two molecules, usually coupled with the hydrolysis of ATP.

   Example: DNA ligase.

Nomenclature

Common names often end with the suffix '-ase' (e.g., hydrolase, isomerase, sucrose $\rightarrow$ sucrase, protein $\rightarrow$ protease). Some older names do not follow this rule (e.g., Pepsin, Trypsin, Renin).

Systematic names are more complex and indicate both the substrate and the type of reaction, following the IUB classification (e.g., Lactate: NAD$^+$ oxidoreductase).

Co-Factors

Some enzymes are composed solely of protein. However, many enzymes require a non-protein component called a cofactor to be catalytically active.

Holoenzyme and Apoenzyme

• Apoenzyme: The protein part of the enzyme. It is catalytically inactive on its own.
• Cofactor: The non-protein part. It is essential for enzyme activity.
• Holoenzyme: The complete, catalytically active enzyme, consisting of the apoenzyme bound to its cofactor.

$ \text{Apoenzyme} + \text{Cofactor} \rightarrow \text{Holoenzyme (Active)} $

Types of Co-factors

Cofactors can be organic or inorganic.

1. Prosthetic groups:
   • These are organic cofactors that are tightly bound to the apoenzyme, often covalently.
   • Example: Heme group in enzyme Catalase and Peroxidase. Heme contains iron ($Fe^{2+}$) which is essential for their activity in breaking down hydrogen peroxide.
2. Coenzymes:
   • These are also organic cofactors, but they are loosely bound to the apoenzyme.
   • They often serve as carriers of atoms or groups (e.g., electron carriers, group carriers).
   • Many coenzymes are derived from vitamins.
   • Example: NAD (Nicotinamide Adenine Dinucleotide) and NADP (Nicotinamide Adenine Dinucleotide Phosphate) are derived from Niacin (Vitamin $B_3$) and are involved in redox reactions as electron carriers. FAD (Flavin Adenine Dinucleotide) is derived from Riboflavin (Vitamin $B_2$). Coenzyme A is derived from Pantothenic acid (Vitamin $B_5$).
3. Metal ions:
   • These are inorganic cofactors.
   • Metal ions form coordinate bonds with side chains at the active site and/or with the substrate.
   • Example: Zinc ($Zn^{2+}$) is a cofactor for carboxypeptidase (a proteolytic enzyme). Magnesium ($Mg^{2+}$) is a cofactor for hexokinase. Molybdenum ($Mo$) is a cofactor for nitrogenase. Copper ($Cu^{2+}$) is a cofactor for tyrosinase.

The presence of a cofactor is crucial for the catalytic activity of many enzymes. If the cofactor is removed, the enzyme becomes inactive.