Enzymes - Comprehensive Study Notes (From Transcript)

CHEMISTRY

  • Introduction

    • Enzymes are biological catalysts that speed up the rate of biochemical reactions.

    • Most enzymes are three-dimensional globular proteins (tertiary and quaternary structure).

    • Some special RNA species act as enzymes and are called ribozymes (e.g., hammerhead ribozyme).

    • Hammerhead enzyme (image/example referenced).

STRUCTURE OF ENZYMES

  • The active site is the region that binds substrates, co-factors, and prosthetic groups and contains residues that help hold the substrate.

  • Active sites generally occupy less than 5% of the total surface area of the enzyme.

  • Active site has a specific shape due to the protein’s tertiary structure.

  • A change in the protein’s shape affects the active site shape and thus enzyme function.

ACTIVE SITE

  • The active site can be divided into:

    • Binding Site: It chooses the substrate and binds it to the active site.

    • Catalytic Site: It performs the catalytic action of the enzyme.

CO-FACTORS

  • Co-factor: a non-protein molecule that enables chemical reactions that cannot be performed by the standard 20 amino acids.

  • Types:

    • Organic co-factors

    • Inorganic cofactors

  • Without the cofactor attached, the protein is not active. Cofactor binding activates the protein.

INORGANIC CO-FACTORS

  • These are inorganic molecules required for proper enzyme activity.

  • Examples:

    • Carbonic anhydrase requires Zn for activity.

    • Hexokinase has co-factor Mg.

ORGANIC CO-FACTORS

  • These are organic molecules required for proper enzyme activity.

  • Example: glycogen phosphorylase requires the small organic molecule pyridoxal phosphate.

TYPES OF ORGANIC CO-FACTORS

  • Prosthetic Group: tightly bound organic co-factor (e.g., flavins, heme groups, biotin).

  • Coenzyme: loosely bound organic co-factor (e.g., NAD+).

  • Structural representations (e.g., NAD+ with nicotinamide, adenine, ribose components).

TYPES OF CO-FACTORS (CONTINUED)

  • An enzyme with its co-factor removed is designated as an apoenzyme.

  • The complete complex of a protein with all necessary small organic molecules, metal ions, and other components is termed a holoenzyme (holoprotein).

  • Terms:

    • Apoenzyme

    • Holoenzyme

SUBSTRATE

  • The reactant in a biochemical reaction is termed the substrate.

  • When a substrate binds to an enzyme, it forms an enzyme–substrate complex.

  • Diagrammatic idea: Substrate joins enzyme to form the complex.

SITES OF ENZYME SYNTHESIS

  • Enzymes are synthesized by ribosomes attached to the rough endoplasmic reticulum.

  • Information for enzyme synthesis is carried by DNA.

  • Amino acids are bonded together to form specific enzymes according to the DNA code.

  • Ribosomes and endoplasmic reticulum participate in this process.

INTRACELLULAR AND EXTRACELLULAR ENZYMES

  • Intracellular enzymes: synthesized and retained inside the cell for cellular use.

    • Found in cytoplasm, nucleus, mitochondria, chloroplasts.

    • Example: Oxidoreductase catalyzes biological oxidation; some enzymes involved in reduction reside in mitochondria.

  • Extracellular enzymes: synthesized in the cell but secreted to function outside the cell.

    • Example: Digestive enzymes produced by the pancreas are transported to the duodenum.

CHARACTERISTICS

  • Enzymes speed up reactions by lowering activation energy.

  • Their presence does not affect the nature or properties of the end product.

  • Enzymes are highly specific; each enzyme catalyzes one kind of substrate.

  • A small amount of enzyme can accelerate chemical reactions.

  • Enzymes are sensitive to changes in pH, temperature, and substrate concentration.

  • Turnover number (Kcat) is the number of substrate molecules transformed per minute by one enzyme molecule.

    • Example: Catalase turnover number = 6×106 min−16 \times 10^{6} \ \text{min}^{-1}

NOMENCLATURE OF ENZYMES

  • An enzyme is named according to the name of the substrate it catalyzes.

  • Historically, some enzymes were named before systematic naming existed (e.g., pepsin, trypsin, rennin).

  • By adding the suffix -ase to the substrate name, enzymes are named (e.g., maltose + water → maltase).

  • Hydrolytic enzymes are termed hydrolases (e.g., maltose + water with maltase).

EXAMPLES

  • Substrate → Enzyme

    • lactose → lactase; products: glucose + galactose

    • maltose → maltase; products: glucose + glucose

    • cellulose → cellulase; products: glucose

    • starch → amylase; products: maltose

    • lipid → lipase; products: glycerol + fatty acids

    • protein → protease; products: peptides + polypeptide

CLASSIFICATION

  • A systematic classification of enzymes has been developed by the International Union of Biochemistry and Molecular Biology (IUBMB).

  • Based on reaction type; six major classes.

  • Each class is subdivided into sub-classes, sub-sub-classes, etc., to describe numerous enzyme-catalyzed reactions.

CLASSIFICATION OF ENZYMES (CONTINUED)

  • ENZYME CLASS – REACTION TYPE – EXAMPLES:

    • Oxidoreductases – Reduction-oxidation (redox) reactions – Example: Lactate dehydrogenase

    • Transferases – Move chemical groups – Example: Hexokinase

    • Hydrolases – Hydrolysis; bond cleavage with transfer of functional group of water – Example: Lysozyme

    • Lyases – Non-hydrolytic bond cleavage – Example: Fumarase

    • Isomerases – Intramolecular group transfer (isomerization) – Example: Triose phosphate isomerase

    • Ligases – Synthesis of new covalent bonds using ATP hydrolysis – Example: RNA polymerase

MECHANISM OF ENZYME ACTION

  • The catalytic efficiency of enzymes is explained from two perspectives:

    • Thermodynamic changes

    • Processes at the active site

THERMODYNAMIC CHANGES

  • All chemical reactions have energy barriers between reactants and products.

  • The difference between the transition state and substrate is called the activational barrier (activation energy).

  • Symbols:

    • Free energy: GG

    • Activation barrier for uncatalyzed: ΔGuncat‡\Delta G^{\ddagger}_{uncat}

    • Activation barrier for catalyzed: ΔGcat‡\Delta G^{\ddagger}_{cat}

  • Diagrammatic idea: Reactants → Transition state (uncatalyzed) → Products; catalyzed path has a lower transition state energy.

  • Enzymes provide an alternate pathway for substrate-to-product conversion, lowering the activation energy and increasing the reaction rate.

  • The total energy of the system remains the same; the equilibrium is not disturbed.

LOCK AND KEY MODEL

  • Proposed by Emil Fischer in 1894.

  • The active site is rigid in shape; there is no change in the active site before or after a reaction.

INDUCED FIT MODEL

  • Proposed by Daniel Koshland in 1958.

  • Substrate binding causes a conformational change in the enzyme; the active site changes shape to accommodate the substrate and bind effectively.

  • Induced Fit Hypothesis: binding induces complementary fit between enzyme and substrate.

PHARMACEUTICAL IMPORTANCE

  • Enzymes are involved in virtually all physiological processes, making them prime targets for drugs.

  • Applied enzyme kinetics is a principal tool for identifying and characterizing therapeutic agents that selectively inhibit specific enzyme-catalyzed processes.

  • Enzyme kinetics plays a critical role in drug discovery and in elucidating the mode of action of drugs.

INHIBITION

  • Inhibition: prevention of an enzyme process due to interaction of inhibitors with the enzyme.

  • Inhibitors: substances that diminish the velocity of an enzyme-catalyzed reaction.

  • Inhibition mechanism often involves the inhibitor blocking the active site.

TYPES OF INHIBITION

  • Reversible Inhibition

  • Irreversible Inhibition

  • Competitive

  • Uncompetitive

  • Mixed

  • Non-competitive

REVERSIBLE INHIBITION

  • Inhibition where the inhibitor can associate and dissociate from the enzyme’s binding site.

  • TYPES (four):

    • Competitive inhibition

    • Uncompetitive inhibition

    • Mixed inhibition

    • Non-competitive inhibition

COMPETITIVE INHIBITION

  • Inhibitors compete with the substrate for the active site.

  • Formation of E.S complex is reduced while a new E.I complex is formed.

  • Substrate cannot bind when inhibitor is bound.

EXAMPLES OF COMPETITIVE INHIBITION

  • Statin drugs (e.g., Lipitor) are competitive inhibitors:

    • They compete with HMG-CoA for the active site of HMG-CoA reductase, inhibiting cholesterol synthesis.

UNCOMPETITIVE INHIBITION

  • The inhibitor does not compete with the substrate for the active site.

  • It binds to a site distinct from the active site (allosteric site).

  • Forms E.S.I complex with the substrate already bound.

EXAMPLES OF UNCOMPETITIVE INHIBITION

  • Drugs used to treat methanol or ethylene glycol poisoning act as uncompetitive inhibitors.

  • Tetramethylene sulfoxide and 3-butylthiolene-1-oxide are uncompetitive inhibitors of liver alcohol dehydrogenase.

MIXED INHIBITION

  • In this type, both E.I and E.S.I complexes are formed.

  • Both complexes are catalytically inactive.

NON-COMPETITIVE INHIBITION

  • A special case of inhibition where the inhibitor has the same affinity for either the free enzyme (E) or the enzyme-substrate complex (E.S).

IRREVERSIBLE INHIBITION

  • The inhibitor covalently attaches to the enzyme, permanently inactivating it.

  • Catalytic activity is completely lost and can only be restored by synthesizing new enzyme.

  • Example: Aspirin covalently modifies a key inflammatory enzyme (irreversible inhibitor).

  • SUICIDE INHIBITION: a special form where the enzyme converts the inhibitor into a reactive form in its active site.

ACTIVATION

  • Activation is the conversion of an inactive form of an enzyme to its active form, enabling metabolic activity.

ACTIVATION TYPES

  • Activation by cofactors.

  • Conversion of an enzyme precursor (zymogen) to its active form.

ACTIVATION BY CO FACTORS

  • Many enzymes are activated by cofactors.

  • Examples:

    • DNA polymerase is a holoenzyme that catalyzes polymerization of deoxyribonucleotides into a DNA strand; it uses Mg2+ for catalytic activity.

    • Horse liver dehydrogenase uses Zn2+ for activation.

DIAGNOSTIC SIGNIFICANCES OF ENZYMES

  • Clinically important enzymes and their concentrations:

    • Lactate dehydrogenase (LDH) 60-120 IU/L

    • Transaminases: AST (SGOT) 5-20 IU/L; ALT (SGPT) 5-15 IU/L

    • Creatine phosphokinase (CPK) 10-60 IU/L

    • Alkaline phosphatase (ALP) 4-17 King-Armstrong units/100 mL

    • Acid phosphatase

    • Isocitrate dehydrogenase

    • Amylase

    • Lipase

  • Concentration increases in: myocardial infarction, myopathy or muscle disorder, leukemias, acute hepatitis, carcinomatosis, liver disorders, pancreatitis, parotitis, obstruction, diabetes, etc.

  • Gamma-glutamyl transpeptidase (GGT): liver damage indicator (alcoholism).

DIAGNOSTIC SIGNIFICANCES OF ISOENZYMES

  • Isoenzymes (isoenzymes) are enzyme variants present in specific tissues and elevated under certain conditions.

  • Examples (LDH isoenzymes):

    • LDH-1: Heart, red blood cells; elevated in myocardial infarction (MI).

    • LDH-2: Heart, red blood cells; associated with kidney disease and megaloblastic anemia.

    • LDH-3: Brain; lung and spleen (tissue distribution varies).

    • LDH-4: Liver and other tissues; describes situations like urea-related conditions.

    • LDH-5: Skeletal muscle and liver; associated with muscle diseases and liver disease.

  • Notes on LDH isoenzyme patterns help in diagnosing specific organ involvement (e.g., MI, liver disease, muscle disorders).

SUMMARY OF KEY EQUATIONS AND NUMBERS

  • Turnover number (Kcat): the number of substrate molecules transformed per minute by one enzyme molecule.

    • Example: Catalase turnover number = 6×106 min−16 \times 10^{6} \ \text{min}^{-1}

  • Activation energy concepts in thermodynamics: ΔG‡<em>uncat>ΔG‡</em>cat\Delta G^{\ddagger}<em>{uncat} > \Delta G^{\ddagger}</em>{cat}, illustrating the lowered barrier when an enzyme is present.

  • Enzyme classes (EC numbers follow formal IUBMB classification, six major classes): Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases.

Note: This set of notes mirrors the content provided in the transcript, organized into clear sections with bullets and key examples, including the major models (Lock and Key; Induced Fit), the inhibition types (competitive, uncompetitive, mixed, non-competitive; reversible vs irreversible; examples like statins and aspirin), and diagnostic implications (enzyme levels and LDH isoenzymes).