Analyzing Chemical Composition in Living Organisms

I. Introduction

• Chemical analysis is essential to understand the composition of living organisms.
• Organic and inorganic compounds contribute to the chemical makeup of living tissues.

II. Performing Chemical Analysis

• Procedure:
  • Take a living tissue (vegetable or liver) and grind it in trichloroacetic acid (Cl3CCOOH) using a mortar and pestle.
  • Obtain a thick slurry.
  • Strain through cheesecloth or cotton, yielding an acid-soluble pool and acid-insoluble fraction.

III. Analysis of Organic Compounds

• Acid-Soluble Pool:
  • Contains thousands of organic compounds.
  • Extract compounds and use separation techniques for isolation and purification.
  • Analytical techniques provide insights into molecular formula and structure.
  • All carbon compounds from living tissues are termed ‘biomolecules.’

IV. Elemental Analysis

• Ash Content:
  • Perform a destructive experiment by weighing and drying a small amount of living tissue.
  • Burning tissue leaves ‘ash’ containing inorganic elements (e.g., calcium, magnesium).
  • Inorganic compounds like sulfate, and phosphate are present in the acid-soluble fraction.
  • Elemental analysis reveals composition in terms of hydrogen, oxygen, chlorine, carbon, etc.

V. Classifying Biomolecules

• From a Biological Perspective:
  • Identify functional groups (aldehydes, ketones, aromatic compounds) from a chemistry viewpoint.
  • Classify into amino acids, nucleotide bases, fatty acids, etc., from a biological standpoint.

VI. Amino Acids

• Definition:
  • Organic compounds with amino and acidic groups on the same α-carbon.
• Structure:
  • Four substituent groups: hydrogen, carboxyl group, amino group, and R group.
• Types:
  • Glycine, alanine, serine, etc., based on the nature of the R group.
• Properties:
  • Acidic, basic, neutral, and aromatic amino acids.
  • Ionizable nature of –NH2 and –COOH groups.

VII. Lipids

• Characteristics:
  • Generally water-insoluble.
  • Include fatty acids, glycerol, and phospholipids.
• Types:
  • Saturated and unsaturated fatty acids.
  • Monoglycerides, diglycerides, triglycerides (fats and oils).
• Phospholipids:
  • Contains phosphorous and phosphorylated organic compounds.
  • Found in cell membranes (e.g., lecithin).

VIII. Heterocyclic Rings and Nucleic Acids

• Compounds with Heterocyclic Rings:
  • Nitrogen bases – adenine, guanine, cytosine, uracil, thymine.
• Nucleosides and Nucleotides:
  • Nucleosides when attached to sugar.
  • Nucleotides when a phosphate group is esterified.
• Nucleic Acids:
  • DNA and RNA consist of nucleotides.
  • Function as genetic material.

Primary and Secondary Metabolites in Living Organisms

I. Primary Metabolites

• Definition:
  • Found in animal tissues.
  • Categories include amino acids, sugars, and others (Figure 9.1).
• Functions:
  • Have identifiable functions.
  • Play known roles in normal physiological processes.

II. Secondary Metabolites

• Definition:
  • Found in plant, fungal, and microbial cells.
  • Include alkaloids, flavonoids, rubber, essential oils, antibiotics, pigments, scents, gums, and spices.
• Characteristics:
  • More diverse than primary metabolites.
  • Often specific to certain organisms.
• Functions:
  • Roles not fully understood.
  • Some contribute to ‘human welfare’ (e.g., rubber, drugs, spices, scents, pigments).
  • Ecological importance, which will be explored in later studies.

III. Significance of Secondary Metabolites

• Human Welfare:
  • Some secondary metabolites are valuable to human needs (rubber, drugs, spices).
• Ecological Importance:
  • Contribute to the ecological dynamics of organisms.
  • Roles may involve interactions with other organisms and the environment.

IV. Future Exploration

• Understanding Roles:
  • Many functions of secondary metabolites are yet to be understood.
  • Ongoing research may reveal additional roles and significance.
• Applications:
  • Potential applications in medicine, agriculture, and industry.
  • Continued study will unveil more about their practical uses.

Biomacromolecules in Living Organisms

I. Molecular Weights of Biomolecules

• Common Feature:
  • Compounds in the acid-soluble pool share molecular weights ranging from 18 to approximately 800 daltons (Da).

II. Acid-Insoluble Fraction

• Composition:
  • Proteins, nucleic acids, polysaccharides, and lipids.
• Molecular Weights:
  • Except for lipids, these compounds have molecular weights in the range of ten thousand daltons and above.
• Classification:
  • Biomolecules are divided into micromolecules (molecular weights < 1000 Da) and macromolecules (found in the acid-insoluble fraction).

III. Macromolecular Nature of Lipids

• Exception:
  • Lipids, despite having molecular weights below 800 Da, are classified as macromolecules.
• Explanation:
  • Lipids are present not only as individual compounds but also form structures like cell membranes.
  • Grinding tissue disrupts cell structures, breaking membranes into vesicles that are not water-soluble.
  • Lipids, in the form of membrane fragments (vesicles), get separated along with the acid-insoluble pool, justifying their macromolecular classification.

IV. Cytoplasmic Composition

• Acid-Soluble Pool:
  • Represents roughly the cytoplasmic composition.
• Macromolecules:
  • Cytoplasmic and organelle macromolecules form the acid-insoluble fraction.
• Representation:
  • Together, they represent the entire chemical composition of living tissues or organisms.

V. Abundance and Class-Wise Arrangement

• Abundance View:
  • Water is the most abundant chemical in living organisms.
• Class-Wise Arrangement:
  • Representing the chemical composition from an abundance perspective reveals the dominance of water in living tissues.

Proteins: Polypeptides and Amino Acid Composition

I. Protein Structure

• Composition:
  • Proteins are polypeptides, consisting of linear chains of amino acids.
  • Amino acids are linked by peptide bonds.

II. Amino Acid Diversity

• Polymer Nature:
  • Each protein is a polymer of amino acids.
  • There are 20 types of amino acids (e.g., alanine, cysteine, proline, tryptophan, lysine, etc.).
• Heteropolymer vs. Homopolymer:
  • Proteins are heteropolymers, containing various types of amino acids.
  • Unlike homopolymers, which consist of a single type of monomer repeating ‘n’ times.

III. Essential Amino Acids

• Importance:
  • Certain amino acids are essential for health, and they must be obtained through the diet.
  • Dietary proteins serve as the source of essential amino acids.
• Classification:
  • Amino acids can be essential or non-essential.
  • Non-essential amino acids are produced by the body, while essential ones must be acquired through diet.

IV. Functions of Proteins

• Diverse Roles:
  • Proteins carry out various functions in living organisms.
  • Examples include nutrient transport across cell membranes, immune response against infectious agents, hormone signaling, enzyme catalysis, and more.

V. Examples of Abundant Proteins

• Collagen:
  • Most abundant protein in the animal world.
• RuBisCO (Ribulose bisphosphate Carboxylase-Oxygenase):
  • Most abundant protein in the entire biosphere.

Polysaccharides: Complex Carbohydrates

I. Polysaccharides in the Acid-Insoluble Pellet

• Class of Macromolecules:
  • Polysaccharides (carbohydrates) are present as another class of macromolecules.

II. Structure and Composition

• Long Chains of Sugars:
  • Polysaccharides are long chains of sugars.
  • They resemble threads, with different monosaccharides as building blocks.
• Examples:
  • Cellulose is a polymeric polysaccharide consisting of glucose monosaccharides.
  • Starch and glycogen are variants of cellulose found in plant and animal tissues, respectively.
  • Inulin is a polymer of fructose.

III. Structural Characteristics

• Homopolymer vs. Variant Structures:
  • Cellulose is a homopolymer, composed of a single type of monosaccharide (glucose).
  • Starch and glycogen are variants of cellulose, serving as energy storehouses in plant and animal tissues.
• Branching:
  • Polysaccharide chains, like glycogen, have a reducing end on the right and a non-reducing end on the left.
  • Branching occurs in the chain structure.

IV. Secondary Structures

• Helical Structures:
  • Starch forms helical secondary structures capable of holding I2 molecules, resulting in a blue color.
  • Cellulose lacks complex helices and cannot hold I2.
• Cellulose in Plant Cell Walls:
  • Plant cell walls are predominantly made of cellulose.
  • Cellulosic materials include paper made from plant pulp and cotton fiber.

V. Complex Polysaccharides

• Building Blocks:
  • Some complex polysaccharides have amino sugars and chemically modified sugars (e.g., glucosamine, N-acetyl galactosamine).
• Example:
  • The exoskeletons of arthropods contain a complex polysaccharide called chitin.
• Homopolymeric Nature:
  • These complex polysaccharides are mostly homopolymers.

Nucleic Acids: Polynucleotides and Nucleotides

I. Nucleic Acids in the Acid-Insoluble Fraction

• Type of Macromolecule:
  • Nucleic acids are the other type of macromolecule found in the acid-insoluble fraction of living tissues.

II. Composition of Macromolecular Fraction

• True Macromolecular Fraction:
  • Polysaccharides, polypeptides, and nucleic acids together constitute the true macromolecular fraction of living tissues or cells.

III. Building Blocks: Nucleotides

• Basic Unit:
  • Nucleic acids are composed of polynucleotides.
  • The building block of nucleic acids is a nucleotide.

IV. Components of Nucleotides

• Three Distinct Components:
  • A nucleotide consists of three chemically distinct components:
    1. Heterocyclic compound
    2. Monosaccharide
    3. Phosphoric acid or phosphate

V. Nitrogenous Bases

• Types:
  • Adenine, guanine, uracil, cytosine, and thymine are the nitrogenous bases found in nucleic acids.
• Classification:
  • Adenine and guanine are substituted purines, while uracil, cytosine, and thymine are substituted pyrimidines.
• Ring Structures:
  • Purine and pyrimidine refer to the skeletal heterocyclic ring structures.

VI. Sugar Component

• Types:
  • The sugar found in polynucleotides can be either ribose (a monosaccharide pentose) or 2’ deoxyribose.
• DNA and RNA:
  • Nucleic acids with deoxyribose are called deoxyribonucleic acid (DNA), and those with ribose are called ribonucleic acid (RNA).

Structure of Proteins: Primary, Secondary, and Tertiary Levels

I. Primary Structure

• Definition:
  • The sequence of amino acids in a protein.
• Representation:
  • Imagined as a linear structure with the N-terminal amino acid on the left and the C-terminal amino acid on the right.
• Importance:
  • The positional information of amino acids along the protein chain.

II. Secondary Structure

• Definition:
  • Regions of the protein chain folded into specific shapes, such as helices.
• Visualization:
  • Helical structures, specifically right-handed helices, are observed.
• Diversity:
  • Only some portions of the protein exhibit helical arrangements.

III. Tertiary Structure

• Definition:
  • The overall three-dimensional folding of the protein.
• Visualization:
  • Folding upon itself, resembling a hollow woolen ball.
• Importance:
  • Essential for the diverse biological activities of proteins.
• Necessary for:
  • Enzyme catalysis, molecular recognition, and other functional aspects.

IV. Specifics of Tertiary Structure

• Folding Patterns:
  • Proteins fold into specific patterns crucial for functionality.
• Biological Activities:
  • Tertiary structure is vital for the diverse biological activities performed by proteins.
  • Enzyme Activity: The specific folding contributes to enzyme activity.
  • Molecular Recognition: Tertiary structure plays a key role in molecular recognition processes.

Enzymes: Nature’s Catalysts

I. Enzymes and Their Nature

• Composition:
  • Almost all enzymes are proteins.
  • Ribozymes, nucleic acids behaving like enzymes, exist as an exception.

II. Structure of Enzymes

• Primary, Secondary, and Tertiary Structure:
  • Enzymes, like proteins, possess primary, secondary, and tertiary structures.
  • Tertiary structure reveals a folded backbone creating crevices or pockets.
• Active Site:
  • Within the tertiary structure, the active site is a crucial pocket where substrates fit.
  • Enzymes catalyze reactions at high rates through their active sites.

III. Contrasting Enzyme Catalysts

• Differences from Inorganic Catalysts:
  • Enzyme catalysts differ from inorganic catalysts in various ways.
  • Notably, enzymes are sensitive to high temperatures, unlike inorganic catalysts.
  • Enzymes from thermophilic organisms display thermal stability even at elevated temperatures (80°-90°C).

IV. Understanding Chemical Reactions

• Types of Changes:
  • Chemical compounds undergo physical and chemical changes.
  • Chemical reactions involve breaking and forming bonds.
• Rate of Processes:
  • Rate is expressed as the amount of product formed per unit time.
  • Rates are influenced by temperature and other factors.
  • Enzyme-catalyzed reactions proceed at significantly higher rates than uncatalyzed ones.

V. Enzyme Power

• Example:
  • Carbonic anhydrase, an enzyme within the cytoplasm, accelerates the formation of H2CO3.
  • Without the enzyme, the reaction is slow, forming about 200 molecules in an hour.
  • With the enzyme, approximately 600,000 molecules are formed every second.
  • Enzymes can accelerate reaction rates by millions of times.

VI. Diversity of Enzymes

• Types:
  • Thousands of enzyme types exist, each catalyzing a unique chemical or metabolic reaction.
• Metabolic Pathways:
  • Multistep chemical reactions, catalyzed by the same enzyme or different enzymes, form metabolic pathways.
  • Example: Glucose → 2 Pyruvic acid involves ten enzyme-catalyzed metabolic reactions.
  • Metabolic pathways lead to diverse end products based on conditions.

Enzyme Catalysis: Overcoming Activation Barriers

I. Active Site Interaction

• Active Site Concept:
  • Enzymes possess three-dimensional structures with active sites.
  • Active sites facilitate the conversion of a substrate (S) into a product (P).

II. Catalytic Process

• Symbolic Representation:
  • Enzyme-catalyzed conversion: �→�S→P.
  • Substrate (S) binds to the enzyme’s active site, forming the ‘ES’ complex.
  • Transient phenomenon: ‘ES’ complex formation.
  • Transition state structure: New structure formed during active site binding.

III. Structural Transformation

• Transition State:
  • The transformation involves the formation of a transition state structure.
  • Intermediate structural states between substrate and product are unstable.
  • Graphical representation: Energy profile (Figure 9.4).

IV. Energy Considerations

• Energy Levels:
  • Energy level difference between ‘S’ and ‘P’ determines reaction exothermicity.
  • Lower ‘P’ level than ‘S’ indicates an exothermic reaction.
• Activation Energy:
  • ‘S’ must go through a higher energy transition state.
  • Difference from the average energy of ‘S’ to the transition state is ‘activation energy.’

V. Enzymatic Facilitation

• Role of Enzymes:
  • Enzymes reduce the activation energy, facilitating the transition of ‘S’ to ‘P.’
  • Overcoming energy barriers makes the reaction more facile.

Nature of Enzyme Action: Catalytic Cycle

I. Enzyme-Substrate Interaction

• Substrate Binding Site:
  • Each enzyme (E) possesses a substrate (S) binding site in its structure.
  • Formation of a highly reactive enzyme-substrate complex (ES).

II. Catalytic Cycle Steps

• Short-Lived Complex:
  • ES complex is short-lived.
  • Dissociation into product(s) (P) and unchanged enzyme.
  • Intermediate formation of the enzyme-product complex (EP).

III. Catalytic Cycle Description

1. Substrate Binding:
   • Substrate (S) binds to the enzyme’s active site.
   • Conformational changes occur in the enzyme.
2. Shape Alteration:
   • Substrate binding induces the enzyme to change its shape.
   • A tighter fit around the substrate is achieved.
3. Chemical Bond Breaking:
   • The active site of the enzyme, now in proximity to the substrate, breaks chemical bonds.
   • Formation of the enzyme-product complex (EP).
4. Product Release:
   • Enzyme releases reaction products.
   • The free enzyme is prepared for the next catalytic cycle.

In summary, the nature of enzyme action involves a catalytic cycle with distinct steps:

1. Substrate binding to the enzyme’s active site.
2. Induced shape alteration in the enzyme for a tighter fit around the substrate.
3. Chemical bonds break in close proximity, leading to the formation of the enzyme-product complex (EP).
4. Release of reaction products, making the enzyme available for subsequent catalytic cycles. This cyclic process ensures efficient and repetitive catalysis.

Factors Affecting Enzyme Activity

• Enzyme activity is influenced by conditions altering the tertiary structure of the protein.
• Factors include temperature, pH, substrate concentration, and specific chemicals.

I. Temperature and pH

• Optimum Conditions:
  • Enzymes have a narrow range of optimal temperature and pH.
  • Optimum temperature and pH: Conditions where enzymes exhibit the highest activity.
• Effect of Temperature:
  • Enzyme activity declines below and above the optimum due to denaturation.
  • Low temperature preserves enzymes temporarily, while high temperature destroys enzymatic activity.

II. Substrate Concentration

• Velocity Increase:
  • Enzymatic reaction velocity initially rises with increasing substrate concentration.
  • Reaches a maximum velocity (Vmax) that is not exceeded with further substrate increase.
  • Saturation point is reached when enzyme molecules are fewer than substrate molecules.

III. Chemical Inhibitors

• Inhibition Process:
  • Enzyme activity sensitive to specific chemicals.
  • Inhibition occurs when a chemical binds to the enzyme and shuts off its activity.
• Competitive Inhibition:
  • Inhibitor closely resembles the substrate in molecular structure.
  • Competes for the substrate binding site.
  • Example: Malonate inhibiting succinic dehydrogenase.

Classification and Nomenclature of Enzymes

1. Oxidoreductases/Dehydrogenases (EC 1)
   • Catalyze oxidoreduction between substrates S and S’.
   • Example: reduced+oxidized′⟶oxidized+reduced
   • S reduced​+S‘oxidized′​⟶S oxidized​+S‘reduced
2. Transferases (EC 2)
   • Catalyze transfer of a group (G, other than hydrogen) between substrates S and S’.
   • Example: S−G+S′⟶S+S′−G
3. Hydrolases (EC 3)
   • Catalyze hydrolysis of ester, ether, peptide, glycosidic, C-C, C-halide, or P-N bonds.
4. Lyases (EC 4)
   • Catalyze removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds.
5. Isomerases (EC 5)
   • Catalyze inter-conversion of optical, geometric, or positional isomers.
6. Ligases (EC 6)
   • Catalyze the linking together of two compounds, e.g., the joining of C-O, C-S, C-N, and P-O bonds.

Co-factors

Enzymes, typically composed of one or several polypeptide chains, may require non-protein components known as cofactors to exhibit catalytic activity. In such cases, the protein part of the enzyme is termed the apoenzyme. Three types of cofactors are identified: prosthetic groups, co-enzymes, and metal ions.

1. Prosthetic Groups
   • Organic compounds are tightly bound to the apoenzyme.
   • Example: Haem in peroxidase and catalase, integral to the enzyme’s active site.
2. Co-enzymes
   • Organic compounds with transient association during catalysis.
   • Serve as co-factors in various enzyme-catalyzed reactions.
   • Often derived from essential vitamins, e.g., nicotinamide adenine dinucleotide (NAD) and NADP contain niacin.
3. Metal Ions
   • Required by some enzymes for activity.
   • Form coordination bonds with side chains at the active site and substrate.
   • Example: Zinc serves as a cofactor for the proteolytic enzyme carboxypeptidase.