Topic 3.1: ENZYMES: Definition, Classification, and Co-factors (1 Hour)

Objective: To define enzymes and describe their fundamental properties as biological catalysts. To learn the systematic IUBMB classification system, which provides a universal language for enzymology. To understand the essential roles of coenzymes and cofactors in enabling and enhancing catalytic activity, with a focus on their relationship with vitamins.

Part 1: Definition and General Properties of Enzymes (20 minutes)

• A. What are Enzymes? The Catalysts of Life

  1. Core Definition: Enzymes are biological catalysts that increase the rate of chemical reactions without being consumed or permanently changed in the process.

  2. Chemical Nature: Nearly all enzymes are globular proteins. Their catalytic activity depends entirely on their specific, native three-dimensional (tertiary and quaternary) structure.

     • The Exception - Ribozymes: A small number of RNA molecules, called ribozymes, also exhibit catalytic activity (e.g., in peptide bond formation on the ribosome). This discovery challenged the "all enzymes are proteins" dogma.

  3. 
     

  4. The Problem Enzymes Solve: The chemical reactions required for life are often very slow under physiological conditions (neutral pH, body temperature of 37°C, atmospheric pressure). Enzymes provide a way to make these reactions occur at a rate compatible with life.

• B. Key Properties of Enzymes:

  1. Catalytic Power: Enzymes are extraordinarily efficient. They can accelerate reaction rates by factors of 10⁶ to 10¹⁴. They do this by lowering the activation energy (Ea) of the reaction.

     • Activation Energy (Ea): The minimum amount of energy required for reactants (substrates) to be converted into products. It can be visualized as an "energy hill" that reactants must climb.

     • Enzymatic Mechanism: An enzyme provides an alternative reaction pathway with a lower "energy hill." It stabilizes the transition state—a high-energy, unstable intermediate state between substrate and product—thereby lowering the activation energy needed to reach it.

     • Important Note: Enzymes do not alter the overall free energy change (ΔG) of the reaction, nor do they change the final equilibrium position (Keq). They only affect the rate at which equilibrium is reached.

  2. High Specificity: Enzymes are highly specific for both the reaction they catalyze and the substrate(s) they act upon. This specificity is a result of the precise three-dimensional structure of the active site.

     • Substrate Specificity: The ability of an enzyme to bind to one or a very limited range of substrates. For example, the enzyme urease acts only on urea.

     • Reaction Specificity: An enzyme typically catalyzes only one specific type of chemical reaction (e.g., oxidation, hydrolysis). There are virtually no side products.

  3. Regulation: Enzyme activity in the cell is not static; it is tightly regulated. This allows metabolic pathways to be turned on or off in response to the cell's needs, maintaining metabolic homeostasis.

  4. Requirement for Mild Conditions: Enzymes function optimally under physiological conditions. Extreme temperatures or pH can cause the protein to denature (lose its native 3D structure), resulting in a loss of catalytic activity.

Part 2: Enzyme Nomenclature and Classification (20 minutes)

A systematic framework is essential for studying the thousands of known enzymes.

• A. Enzyme Nomenclature:

  1. Common/Trivial Names: These are often short, convenient names.

     • May end in the suffix "-ase" attached to the name of the substrate (e.g., Urease acts on urea; Lactase acts on lactose).

     • May describe the function of the enzyme (e.g., Lactate Dehydrogenase removes hydrogen from lactate).

     • Some historical names give no hint of function or substrate (e.g., Pepsin, Trypsin).

  2. Systematic Name (IUBMB System):

     • The International Union of Biochemistry and Molecular Biology (IUBMB) developed a system for unambiguous naming.

     • The systematic name describes the substrate(s), the coenzyme (if any), and the type of reaction catalyzed.

     • Enzyme Commission (EC) Number: Each enzyme is assigned a unique four-digit classification number (e.g., EC 2.7.1.1 for Hexokinase).

       • 1st digit: Major class.

       • 2nd digit: Subclass.

       • 3rd digit: Sub-subclass.

       • 4th digit: Serial number of the specific enzyme.

• B. The Six Major Classes of Enzymes ():**
  (Mnemonic: OTH LIL or Over The Hill, LIL)

  1. EC 1: Oxidoreductases:

     • Function: Catalyze oxidation-reduction (redox) reactions; the transfer of electrons or hydrogen atoms.

     • Common Subclass Names: Dehydrogenase, Oxidase, Reductase, Peroxidase, Hydroxylase.

     • Example: Lactate dehydrogenase (EC 1.1.1.27) oxidizes lactate to pyruvate.

• 2. EC 2: Transferases:

     • Function: Catalyze the transfer of a functional group (other than hydrogen) from a donor molecule to an acceptor molecule.

     • Common Subclass Names: Transaminase (or Aminotransferase), Kinase, Methyltransferase.

     • Example: Hexokinase (EC 2.7.1.1) transfers a phosphate group from ATP to glucose.

  3. EC 3: Hydrolases:

     • Function: Catalyze the cleavage (hydrolysis) of a bond by the addition of a water molecule. This class includes most digestive enzymes.

     • Common Subclass Names: Peptidase, Esterase, Glycosidase, Phosphatase.

     • Example: Acetylcholinesterase (EC 3.1.1.7) hydrolyzes acetylcholine.

  4. EC 4: Lyases:

     • Function: Catalyze the cleavage of C-C, C-S, and C-N bonds by mechanisms other than hydrolysis or oxidation, often resulting in the formation of a double bond or a ring structure.

     • Common Subclass Names: Decarboxylase, Aldolase, Synthase*.

     • Example: Aldolase (EC 4.1.2.13) cleaves fructose-1,6-bisphosphate in glycolysis.

     • *Note: The name "synthase" is reserved for lyases, while "synthetase" is used for ligases (which use ATP).

  5. EC 5: Isomerases:

     • Function: Catalyze geometric or structural rearrangements within a single molecule, interconverting isomers.

     • Common Subclass Names: Isomerase, Mutase, Epimerase, Racemase.

     • Example: Phosphoglucose isomerase (EC 5.3.1.9) converts glucose-6-phosphate to fructose-6-phosphate.

  6. EC 6: Ligases:

     • Function: Catalyze the joining (ligation) of two molecules, a reaction that is energetically unfavorable and is therefore coupled to the hydrolysis of a high-energy phosphate bond (usually from ATP).

     • Common Subclass Names: Synthetase, Carboxylase.

     • Example: Pyruvate carboxylase (EC 6.4.1.1) joins CO₂ to pyruvate, using ATP.

Part 3: Cofactors and Coenzymes - The "Helper" Molecules (20 minutes)

Many enzymes cannot function alone and require a non-protein "helper" molecule to carry out their catalytic function.

• A. Essential Terminology:

  1. Apoenzyme: The inactive protein component of an enzyme.

  2. Cofactor: The non-protein component required for enzyme activity.

  3. Holoenzyme: The complete, catalytically active enzyme, consisting of the apoenzyme plus its cofactor.
     Holoenzyme = Apoenzyme + Cofactor

• B. Types of Cofactors:

  1. Essential Metal Ions (Inorganic Cofactors):

     • Also known as enzyme activators.

     • They may be loosely bound or tightly integrated into the enzyme structure (as in metalloenzymes).

     • Roles:

       • Can form a coordinate bond with the substrate to orient it correctly in the active site.

       • Can stabilize negative charges that develop during the reaction.

       • Can participate directly in redox reactions by changing their oxidation state.

     • Examples:

       • Zinc (Zn²⁺): A cofactor for Carbonic Anhydrase and Alcohol Dehydrogenase.

       • Magnesium (Mg²⁺): Essential for all enzymes that use ATP (e.g., all kinases), where it stabilizes the negative charges on the phosphate groups.

       • Iron (Fe²⁺ or Fe³⁺): Found in the heme group of cytochromes and catalase.

       • Copper (Cu²⁺): A cofactor for Cytochrome C Oxidase.

  2. Coenzymes (Organic Cofactors):

     • Definition: Small, organic, non-protein molecules that act as transient carriers of specific atoms or functional groups. They are often called the "second substrate."

     • The Vitamin Connection: Most coenzymes are synthesized from water-soluble B-complex vitamins. A deficiency of a B vitamin often manifests as a disease because the activity of enzymes requiring that coenzyme is impaired.

     • Classification based on binding:

       • Cosubstrates: Loosely bound to the enzyme. They bind to the active site, are chemically modified during the reaction, and then dissociate. They must be regenerated in a separate reaction. NAD⁺ is the classic example.

       • Prosthetic Groups: Tightly, often covalently, bound to the enzyme. They remain attached to the enzyme throughout the catalytic cycle and are regenerated as part of the cycle. FAD and Heme are classic examples.

• C. Important Coenzymes and their Vitamin Precursors (Clinically Essential to Know):

Coenzyme

Vitamin Precursor

Functional Group Transferred

Key Role/Reaction

NAD⁺, NADP⁺

Niacin (B3)

Hydride ion (H⁻)

Redox reactions (Dehydrogenases)

FAD, FMN

Riboflavin (B2)

Hydrogen atoms (2H)

Redox reactions (Dehydrogenases)

Thiamine Pyrophosphate (TPP)

Thiamine (B1)

Aldehyde group

Oxidative decarboxylation (e.g., PDH)

Coenzyme A (CoA)

Pantothenic Acid (B5)

Acyl group

Acyl group transfer (e.g., Citric Acid Cycle)

Pyridoxal Phosphate (PLP)

Pyridoxine (B6)

Amino group

Transamination, Decarboxylation of AAs

Biotin

Biotin (B7)

Carbon dioxide (CO₂)

Carboxylation reactions (e.g., Pyruvate Carboxylase)

Tetrahydrofolate (THF)

Folic Acid (B9)

One-carbon units

Synthesis of purines and thymine

Cobalamin (B12)

Cobalamin (B12)

Methyl group, H atoms

Methylation (Methionine synthesis)

Topic 3.2: Mechanism of Enzyme Action and Enzyme Kinetics (1 Hour)

Objective: To understand the molecular mechanisms by which enzymes accelerate reaction rates, focusing on the active site and the transition state. To describe the factors that influence the velocity of enzyme-catalyzed reactions and to master the Michaelis-Menten model of enzyme kinetics, including the significance of Vmax and Km.

Part 1: Mechanism of Enzyme Action (20 minutes)

• A. The Concept of Activation Energy and the Transition State:

  1. For any chemical reaction S → P, the substrate (S) does not instantly become the product (P). It must first pass through a high-energy, unstable intermediate state called the transition state (S‡).

  2. Activation Energy (Ea): The amount of energy required to raise the substrate from its ground state to the energy level of the transition state. It is the "energy barrier" to the reaction.

  3. How Enzymes Work: Enzymes do not change the starting or ending energy levels (the ΔG of the reaction). Instead, they provide an alternative reaction pathway with a significantly lower activation energy. They do this by binding to the substrate and stabilizing the transition state. A lower energy barrier means that at any given time, a much larger fraction of substrate molecules will have enough energy to overcome the barrier, leading to a dramatic increase in the reaction rate.

• B. The Active Site (): The Heart of the Enzyme**

  1. Definition: The active site is a specific, three-dimensional cleft or pocket on the surface of the enzyme. It is formed by a small number of amino acid R-groups (the catalytic residues) that are brought together by the protein's tertiary structure.

  2. Function: The active site has two primary functions:

     • Binding Site: It binds the substrate(s) with high specificity and in the correct orientation for catalysis.

     • Catalytic Site: It contains the residues that directly participate in the chemical reaction, facilitating bond breaking and bond making.

• C. Models of Enzyme-Substrate Interaction:

  1. Lock and Key Model (Emil Fischer, 1894):

     • Concept: This early model proposed that the active site has a rigid shape that is perfectly complementary to the shape of the substrate, like a key fitting into a lock.

     • Limitation: This model is too static and does not explain how the enzyme stabilizes the transition state.

  2. Induced Fit Model (Daniel Koshland, 1958):

     • Concept: This is the more accepted model. It proposes that the active site is flexible. The initial binding of the substrate is not perfect. The binding itself induces a conformational change in the enzyme, causing the active site to mold itself around the substrate for a more precise fit.

     • Significance: This induced fit serves two purposes:

       • It aligns the catalytic groups of the enzyme perfectly with the bonds to be altered in the substrate.

       • The binding process itself can strain or distort the substrate's bonds, pushing it closer to the transition state's conformation, thus lowering the activation energy.

Part 2: Factors Affecting the Rate of Enzyme-Catalyzed Reactions (15 minutes)

The velocity (or rate) of an enzyme-catalyzed reaction is influenced by several key factors.

• A. Effect of Enzyme Concentration [E]:

  • If the substrate concentration [S] is kept constant and in excess, the initial reaction velocity (v₀) is directly proportional to the concentration of the enzyme.

  • Rationale: Doubling the amount of enzyme doubles the number of active sites available, thus doubling the reaction rate.

  • Clinical Application: This linear relationship is the basis for most clinical enzyme assays. By ensuring all other factors are optimal and non-limiting, the measured rate of reaction in a patient's serum is directly proportional to the amount of that enzyme present.

• B. Effect of Temperature:

  • Initial Increase: As temperature increases from a low value, the velocity of the reaction increases. This is because both the enzyme and substrate molecules have more kinetic energy, leading to more frequent and energetic collisions.

  • Optimum Temperature: The velocity is maximal at the enzyme's optimum temperature. For most human enzymes, this is around 37°C.

  • Rapid Decrease: At temperatures above the optimum, the enzyme's structure begins to break down. The increased thermal energy disrupts the weak non-covalent bonds that maintain the tertiary structure, causing the enzyme to denature. This leads to a rapid loss of active site conformation and a sharp drop in activity.

  • Clinical Note: High fevers (>40°C) can be dangerous because they can begin to denature critical metabolic enzymes.

• C. Effect of pH:

  • Optimum pH: Each enzyme has a characteristic optimum pH at which its activity is maximal.

  • Mechanism: Changes in pH alter the ionization state of the amino acid R-groups, particularly those in the active site. For an enzyme to be active, its catalytic residues must be in the correct protonation state (e.g., a histidine may need to be protonated to act as an acid, while a glutamate may need to be deprotonated to act as a base).

  • Extreme pH: Drastic changes in pH away from the optimum can lead to complete denaturation of the enzyme by disrupting the ionic bonds that maintain its structure.

  • Physiological Examples:

    • Pepsin (in the stomach): Optimum pH ≈ 1.5 - 2.5

    • Trypsin (in the small intestine): Optimum pH ≈ 8.0

    • Most intracellular enzymes: Optimum pH ≈ 7.4

Part 3: Enzyme Kinetics - The Michaelis-Menten Model (25 minutes)

Enzyme kinetics is the quantitative study of the rates of enzyme-catalyzed reactions.

• A. Effect of Substrate Concentration [S]:

  • If we fix the [E], temperature, and pH, and measure the initial velocity (v₀) at various substrate concentrations [S], we get a characteristic hyperbolic curve.

  • Analysis of the Curve:

    • At low [S]: The velocity is directly proportional to the substrate concentration (v₀ ∝ [S]). This is first-order kinetics because the rate is limited by how often the substrate collides with an empty active site.

    • At high [S]: The velocity becomes constant and independent of the substrate concentration. It approaches a maximum value, Vmax. This is zero-order kinetics because the enzyme's active sites are saturated with substrate. The rate is now limited only by how fast the enzyme can process the substrate and release the product.

• B. The Michaelis-Menten Equation and its Parameters:

  • Leonor Michaelis and Maud Menten proposed a simple model to explain this hyperbolic relationship.

  • The Equation: v₀ = (Vmax * [S]) / (Km + [S])

  • Vmax (Maximum Velocity):

    • Definition: The theoretical maximum rate of the reaction when the enzyme is fully saturated with substrate.

    • Significance: Vmax is directly proportional to the enzyme concentration [E]. Vmax = k_cat * [E]total, where k_cat is the turnover number (the number of substrate molecules one enzyme molecule can convert per second).

  • Km (The Michaelis Constant):

    • Operational Definition: Km is the substrate concentration [S] at which the initial reaction velocity (v₀) is exactly half of Vmax (i.e., when v₀ = ½ Vmax).

    • Significance - An Inverse Measure of Affinity: Km is often used as a measure of the affinity of an enzyme for its substrate.

      • A low Km value indicates a high affinity. This means the enzyme requires only a low concentration of substrate to become half-saturated. It "binds tightly."

      • A high Km value indicates a low affinity. The enzyme requires a high concentration of substrate to become half-saturated. It "binds weakly."

    • Clinical Example: Glucokinase vs. Hexokinase. Both enzymes phosphorylate glucose. Hexokinase (in most tissues) has a low Km for glucose (~0.1 mM), so it is active even at low blood glucose levels. Glucokinase (in liver and pancreas) has a high Km (~10 mM), so it is only significantly active when blood glucose is high (e.g., after a meal), allowing the liver to efficiently take up and store excess glucose.

• C. The Lineweaver-Burk Plot (Double-Reciprocal Plot):

  • Problem: It is difficult to determine Vmax accurately from the hyperbolic Michaelis-Menten plot because the curve approaches Vmax asymptotically.

  • Solution: The Michaelis-Menten equation can be algebraically rearranged into a linear form by taking the reciprocal of both sides. This yields the Lineweaver-Burk equation:
    1/v₀ = (Km/Vmax) * (1/[S]) + 1/Vmax

  • The Plot: This is a plot of 1/v₀ (on the y-axis) versus 1/[S] (on the x-axis), which gives a straight line.

  • Interpreting the Plot: This plot provides a much more accurate way to determine Vmax and Km.

    • Y-intercept = 1/Vmax

    • X-intercept = -1/Km

    • Slope = Km/Vmax

  • Significance: The Lineweaver-Burk plot is particularly useful for analyzing the mechanisms of enzyme inhibition, as different types of inhibitors produce distinct changes in the plot's intercepts and slope.

Topics 3.3 & 3.4: Enzyme Inhibition (2 Hours)

Objective: To define and classify different types of enzyme inhibitors based on their mechanism of action and reversibility. To understand and be able to predict the effects of each type of inhibition on the kinetic parameters Vmax and Km. To appreciate the profound clinical and pharmacological relevance of enzyme inhibition in drug therapy and toxicology.

Part 1: Introduction to Enzyme Inhibition & Reversible Inhibition (Competitive) (1 Hour)

• A. What is Enzyme Inhibition?

  • Definition: An enzyme inhibitor is any substance that binds to an enzyme and decreases its catalytic activity.

  • Significance:

    1. Pharmacology: Most drugs are enzyme inhibitors. By inhibiting a key enzyme in a pathogen's metabolic pathway or a dysregulated human pathway, diseases can be treated.

    2. Toxicology: Many poisons and toxins act by inhibiting critical enzymes (e.g., cyanide inhibits cytochrome c oxidase).

    3. Metabolic Regulation: Natural inhibitors are a key part of cellular metabolic control (e.g., feedback inhibition).

    4. Research: Inhibitors are invaluable tools for studying enzyme mechanisms and metabolic pathways.

• B. Classification of Inhibition:
  Inhibition is broadly classified into two main categories based on whether the inhibitor's effect can be reversed.

  • Reversible Inhibition: The inhibitor binds to the enzyme via non-covalent bonds (H-bonds, ionic bonds, hydrophobic interactions). The inhibitor-enzyme complex can readily dissociate, restoring enzyme activity. E + I ⇌ EI.

  • Irreversible Inhibition: The inhibitor binds very tightly, often covalently, to the enzyme. The dissociation of the inhibitor is extremely slow or non-existent, leading to permanent inactivation of the enzyme. E + I → E-I.

• C. Reversible Inhibition: Competitive Inhibition (* - Most Important Type)**

  • Mechanism:

    1. The inhibitor molecule is a structural analog of the normal substrate. It has a chemical structure that is similar enough to the substrate to be able to fit into the enzyme's active site.

    2. The inhibitor and the substrate are in direct competition for binding to the same active site.

    3. Binding is mutually exclusive: either the substrate can bind, or the inhibitor can bind, but not both at the same time.

    4. E + S ⇌ ES → E + P

    5. E + I ⇌ EI (inactive complex)

  • Overcoming the Inhibition:

    1. Since the binding is reversible and competitive, the effect of the inhibitor can be overcome by sufficiently increasing the substrate concentration [S].

    2. At a very high [S], the substrate molecules will vastly outnumber the inhibitor molecules, effectively "swamping out" the inhibitor and ensuring that the active sites are almost always occupied by the substrate.

  • Effect on Enzyme Kinetics (Vmax and Km):

    1. Effect on Vmax: Vmax remains unchanged.

       • Reasoning: At an infinitely high substrate concentration, the substrate will successfully out-compete the inhibitor for all active sites. Therefore, the reaction can eventually reach the same maximum velocity as it would in the absence of the inhibitor.

    2. Effect on Km: Km is increased.

       • Reasoning: Km is the substrate concentration needed to achieve ½ Vmax. In the presence of a competitive inhibitor, more substrate is required to "fight off" the inhibitor and achieve that half-maximal rate. Therefore, the apparent Km (Km,app) is higher than the true Km. The degree of increase depends on the inhibitor's concentration and its affinity for the enzyme.

• • Lineweaver-Burk Plot for Competitive Inhibition:

    1. The plot shows two lines that intersect on the Y-axis.

    2. Y-intercept (1/Vmax): Unchanged. Both lines have the same Y-intercept.

    3. X-intercept (-1/Km): Changed. The line with the inhibitor is shifted to the right, closer to zero, reflecting a larger Km,app (since -1/Km,app is a smaller negative number).

    4. Slope (Km/Vmax): Increased.

  • Classic Pharmacological Examples:

    1. Statins (e.g., Atorvastatin, Simvastatin) for Hypercholesterolemia:

       • Target Enzyme: HMG-CoA Reductase, the rate-limiting enzyme in cholesterol synthesis.

       • Mechanism: The statin drugs are structural analogs of the HMG-CoA substrate and act as potent competitive inhibitors of the enzyme, thereby lowering cholesterol production.

    2. Methanol Poisoning Treatment:

       • Problem: Methanol is metabolized by alcohol dehydrogenase to toxic formaldehyde and formic acid, which cause blindness and metabolic acidosis.

       • Treatment: Ethanol is administered intravenously. Ethanol is also a substrate for alcohol dehydrogenase and has a much higher affinity (lower Km) for the enzyme. It acts as a competitive inhibitor, preventing the metabolism of methanol to its toxic products. This allows the unmetabolized methanol to be slowly excreted by the kidneys. Fomepizole is another competitive inhibitor used for this purpose.

Part 2: Other Forms of Inhibition (Non-competitive, Uncompetitive, Irreversible) (1 Hour)

• A. Reversible Inhibition: Non-competitive Inhibition ()*

  • Mechanism:

    1. The inhibitor has no structural similarity to the substrate.

    2. It binds to the enzyme at a site different from the active site (an allosteric site).

    3. The inhibitor can bind to either the free enzyme (E) or the enzyme-substrate complex (ES) with equal affinity.

    4. Binding of the inhibitor causes a conformational change that inactivates the catalytic site, but it does not prevent the substrate from binding to the active site.

    5. E + S ⇌ ES → E + P

    6. E + I ⇌ EI (inactive)

    7. ES + I ⇌ ESI (inactive)

  • Overcoming the Inhibition:

    1. This type of inhibition cannot be overcome by increasing the substrate concentration. The inhibitor is not competing for the active site. Once an enzyme molecule is bound by the inhibitor, it is effectively "poisoned" and removed from the pool of active enzymes, regardless of how much substrate is present.

  • Effect on Enzyme Kinetics (Vmax and Km):

    1. Effect on Vmax: Vmax is decreased.

       • Reasoning: The inhibitor effectively reduces the concentration of active enzyme molecules available for catalysis. Since Vmax is proportional to the concentration of active enzyme, the maximum achievable velocity is lowered.

    2. Effect on Km: Km remains unchanged.

       • Reasoning: The inhibitor does not interfere with the binding of the substrate to the active site of the enzyme molecules that are not bound by the inhibitor. Therefore, the affinity of the substrate for the active enzyme is unchanged.

  • Lineweaver-Burk Plot for Non-competitive Inhibition:

    1. The plot shows two lines with different Y-intercepts that intersect on the X-axis.

    2. Y-intercept (1/Vmax): Increased (since Vmax is decreased).

    3. X-intercept (-1/Km): Unchanged. Both lines have the same X-intercept.

    4. Slope (Km/Vmax): Increased.

  • Examples:

    1. Heavy metal poisoning: Ions like Lead (Pb²⁺) and Mercury (Hg²⁺) are non-competitive inhibitors. They have a high affinity for the sulfhydryl (-SH) groups of cysteine residues in enzymes, disrupting their structure and catalytic function.

• B. Reversible Inhibition: Uncompetitive Inhibition

  • Mechanism:

    1. A less common type of inhibition.

    2. The inhibitor can bind only to the enzyme-substrate (ES) complex, not to the free enzyme. The binding of the substrate creates or exposes the binding site for the inhibitor.

    3. ES + I ⇌ ESI (inactive)

  • Effect on Enzyme Kinetics:

    1. Vmax is decreased. The formation of the ESI complex removes ES from the reaction pathway.

    2. Km is also decreased. The inhibitor effectively "siphons off" the ES complex, which, by Le Châtelier's principle, pulls the E + S ⇌ ES equilibrium to the right, increasing the apparent affinity of the enzyme for the substrate.

  • Lineweaver-Burk Plot: The plot shows two parallel lines with different intercepts.

• C. Irreversible Inhibition (): Permanent Inactivation**

  • Mechanism: The inhibitor forms a very stable, often covalent, bond with a functional group in the enzyme's active site. The enzyme is permanently inactivated. The cell must synthesize new enzyme molecules to restore activity.

  • Examples:

    1. Organophosphates: Found in some insecticides and nerve gases (e.g., Sarin). They are irreversible inhibitors of acetylcholinesterase. Inhibition of this enzyme leads to the accumulation of the neurotransmitter acetylcholine, causing overstimulation of the nervous system and paralysis.

    2. Heavy Metals: Lead and mercury can also act as irreversible inhibitors by forming tight covalent bonds with -SH groups.

• D. Suicide Inhibition: A Special Case of Irreversible Inhibition ():*

  • Mechanism: Also known as mechanism-based inactivation. This is a highly specific and potent form of irreversible inhibition.

    1. The inhibitor is designed to be a substrate for the target enzyme.

    2. The enzyme begins to process the inhibitor as if it were the normal substrate.

    3. During the catalytic process, the enzyme itself converts the inhibitor into a highly reactive intermediate.

    4. This reactive intermediate does not leave the active site; instead, it immediately forms a covalent bond with a key amino acid residue, permanently inactivating the enzyme. The enzyme essentially "commits suicide" by processing the inhibitor.

  • Pharmacological Significance: Suicide inhibitors are often very specific and potent drugs because they are only activated by their target enzyme.

  • Classic Examples:

    1. Allopurinol: Used to treat gout. Gout is caused by an overproduction of uric acid. Xanthine oxidase is the enzyme that produces uric acid. Allopurinol is a substrate for xanthine oxidase, which converts it to alloxanthine. Alloxanthine is a potent inhibitor that remains tightly bound to the enzyme's active site, inactivating it.

    2. Aspirin (Acetylsalicylic Acid): Aspirin is a suicide inhibitor of the enzyme cyclooxygenase (COX). Aspirin acetylates a serine residue in the active site of COX, irreversibly blocking the synthesis of prostaglandins, which are mediators of pain, inflammation, and fever.

Topic 3.5: Enzyme Regulation (1 Hour)

Objective: To understand the various physiological mechanisms by which the activity of enzymes is controlled within the cell. This includes rapid, short-term regulation (allosteric control, covalent modification) and slow, long-term regulation (control of enzyme synthesis). A deep understanding of these processes is fundamental to appreciating metabolic homeostasis and how it is disrupted in disease.

Part 1: The Need for Regulation and Allosteric Control (25 minutes)

• A. Why Regulate Enzymes?

  • Cells live in a constantly changing environment and must be able to adapt their metabolic activity accordingly.

  • Metabolic Homeostasis: Regulation allows the cell to maintain a stable internal environment by balancing the rates of anabolic (synthesis) and catabolic (breakdown) pathways.

  • Efficiency: It prevents the wasteful production of metabolic intermediates when they are not needed and ensures that energy is generated only when required.

  • Coordination: It allows for the coordinated operation of complex, interconnected metabolic pathways.

  • Primary Target for Regulation: The activity of the rate-limiting enzyme of a pathway is almost always the main point of control. This is typically the first irreversible, committed step of the pathway. Regulating this step is the most efficient way to control the overall flux through the entire pathway.

• B. Allosteric Regulation: The Primary Rapid Response Mechanism ()*

  • Definition: Allosteric regulation is the control of an enzyme's activity by the binding of a regulatory molecule (a modulator or effector) to a site on the enzyme that is topographically distinct from the active site. This site is called the allosteric site (from Greek allos, "other" + stereos, "space").

  • Mechanism:

    • The binding of the modulator to the allosteric site is reversible and non-covalent.

    • This binding triggers a rapid conformational change in the enzyme's structure.

    • This conformational change is transmitted to the active site, altering its shape and, consequently, its affinity for the substrate and/or its catalytic activity.

  • Types of Allosteric Modulators:

    • Positive Modulator (Allosteric Activator): Binds to the allosteric site and increases the enzyme's activity. It often stabilizes the enzyme in a high-affinity "R" (relaxed) state.

    • Negative Modulator (Allosteric Inhibitor): Binds to the allosteric site and decreases the enzyme's activity. It often stabilizes the enzyme in a low-affinity "T" (tense) state.

  • Properties of Allosteric Enzymes:

    • They are generally larger and more complex than non-allosteric enzymes, often consisting of multiple subunits (they have quaternary structure).

    • They exhibit cooperativity. The binding of a substrate molecule to one active site can influence the binding of substrate to other active sites on the same enzyme molecule.

    • They do not follow Michaelis-Menten kinetics. Instead, they display a sigmoidal (S-shaped) curve when plotting reaction velocity versus substrate concentration. This allows them to be very sensitive to small changes in substrate concentration, acting like metabolic "switches."

• C. Feedback Inhibition: A Key Type of Allosteric Regulation

  • Definition: A common regulatory strategy where the final end-product of a metabolic pathway acts as an allosteric inhibitor of an enzyme that catalyzes an early, committed step in that same pathway.

  • Physiological Significance: This creates an elegant and efficient negative feedback loop.

    • When the end-product accumulates to a sufficient level, it "shuts down" its own synthesis by inhibiting the first enzyme, preventing wasteful overproduction.

    • When the cell consumes the end-product and its concentration falls, the inhibition is relieved, and the pathway becomes active again.

  • Example: Synthesis of CTP (a pyrimidine nucleotide). The final product, CTP, is an allosteric inhibitor of the first enzyme in the pathway, Aspartate Transcarbamoylase (ATCase). ATP, a purine nucleotide, acts as an allosteric activator, helping to balance the synthesis of purines and pyrimidines.

Part 2: Other Rapid and Long-Term Regulatory Mechanisms (25 minutes)

• A. Covalent Modification: The "On/Off" Switch

  • Definition: The activity of an enzyme is rapidly altered by the covalent attachment or removal of a chemical group to one or more of its amino acid residues.

  • Reversible Phosphorylation and Dephosphorylation: The Most Common Mechanism:

    • This is the major mechanism for regulating intracellular processes in response to extracellular signals (e.g., hormones like insulin and glucagon).

    • Protein Kinases: Enzymes that catalyze the transfer of the terminal phosphate group from ATP to a specific serine, threonine, or tyrosine residue on a target enzyme (phosphorylation).

    • Protein Phosphatases: Enzymes that catalyze the removal of this phosphate group by hydrolysis (dephosphorylation).

    • Effect: The addition of the bulky, negatively charged phosphate group causes a significant conformational change, which can either activate or inhibit the enzyme, depending on the specific protein.

    • Classic Example - Glycogen Metabolism:

      • Glycogen Phosphorylase (breaks down glycogen): Is activated by phosphorylation (in response to glucagon or adrenaline).

      • Glycogen Synthase (synthesizes glycogen): Is inactivated by phosphorylation.

      • This reciprocal regulation ensures that the two opposing pathways are not active at the same time, preventing a futile cycle.

• B. Zymogen Activation (Irreversible Proteolytic Cleavage):

  • Definition: Some enzymes, particularly those that could cause damage if active in their site of synthesis (e.g., potent proteases), are synthesized and secreted as inactive, larger precursors called zymogens or proenzymes.

  • Activation Mechanism: They are activated by the irreversible hydrolytic cleavage of one or more specific peptide bonds by a protease. This cleavage removes an inhibitory peptide segment, causing a conformational change that unmasks the active site.

  • Significance: This provides a mechanism for safely storing and transporting potent enzymes, ensuring they are activated only at the specific time and place they are needed.

  • Key Examples:

    • Digestive Enzymes:

      • In the stomach, pepsinogen is activated to pepsin by the acidic pH and by pepsin itself (auto-activation).

      • In the small intestine, pancreatic trypsinogen is activated to trypsin by the enzyme enteropeptidase. Trypsin then activates all other pancreatic zymogens (chymotrypsinogen, proelastase, procarboxypeptidases).

    • Blood Clotting Cascade: The formation of a blood clot involves a cascade of sequential zymogen activations, where each activated protease activates the next zymogen in the series (e.g., prothrombin is cleaved to form active thrombin). This allows for rapid and massive amplification of the initial signal.

• C. Induction and Repression of Enzyme Synthesis: Long-Term Adaptation

  • The regulatory mechanisms discussed so far (allosteric, covalent) are rapid and alter the activity of existing enzyme molecules. In contrast, this mechanism regulates the total amount of enzyme protein in the cell.

  • Definition: This is a slower, long-term adaptive response that involves changing the rate of enzyme synthesis at the level of gene expression (transcription and translation).

  • Induction: The presence of a specific molecule (an inducer, often a substrate) increases the synthesis of the enzyme(s) required to metabolize it.

    • Example: High levels of dietary fats can induce the synthesis of enzymes involved in fatty acid oxidation.

  • Repression: The presence of a specific molecule (a repressor, often an end-product) decreases the synthesis of the enzymes in its own biosynthetic pathway.

    • Example: The presence of cholesterol in the diet represses the synthesis of HMG-CoA reductase, the key enzyme for cholesterol biosynthesis.

Summary of Regulatory Strategies (10 minutes):

Mechanism

Speed

Reversibility

How it Works

Example

Allosteric Control

Very Fast (sec)

Reversible

Modulator binds to allosteric site, changing active site conformation

Feedback inhibition of ATCase by CTP

Covalent Modification

Fast (min)

Reversible

Phosphorylation/dephosphorylation alters enzyme activity

Activation of glycogen phosphorylase

Zymogen Activation

Fast (min)

Irreversible

Proteolytic cleavage of an inactive precursor

Activation of trypsinogen to trypsin

Enzyme Synthesis

Slow (hours/days)

Reversible

Altering gene expression to change the amount of enzyme

Induction of metabolic enzymes by hormones

Conclusion: The cell employs a sophisticated hierarchy of regulatory strategies to control its enzymatic machinery. Rapid, reversible mechanisms like allosteric control and phosphorylation allow for minute-to-minute adjustments to metabolic flux, while slower, long-term control of enzyme synthesis allows for adaptation to more sustained changes in the cellular environment or nutritional state. The failure of these regulatory systems is a hallmark of many disease states, including cancer and metabolic disorders.

Topics 3.6 & 3.7: Clinical Enzymology (1 Hour)

Objective: To understand the principles behind using enzyme measurements in plasma or serum as diagnostic and prognostic tools. To differentiate between functional and non-functional plasma enzymes and to identify the key organ-specific enzymes and isoenzymes used to diagnose damage to the heart, liver, pancreas, and other tissues.

Part 1: Principles of Diagnostic Enzymology (15 minutes)

• A. The Concept of Plasma Enzymes:

  • The enzymes found in plasma can be divided into two major groups:

    1. Functional Plasma Enzymes:

       • Definition: These are enzymes that are actively synthesized in an organ (like the liver) and are deliberately secreted into the plasma, where they perform a specific physiological function.

       • Characteristics: Their concentration is normally high in the plasma compared to the tissues where they are made.

       • Clinical Significance: A decrease in their plasma levels often indicates a disease of the organ that synthesizes them (e.g., severe liver disease).

       • Examples:

         • Enzymes of the Blood Coagulation Cascade: Thrombin, Factor Xa, etc., synthesized by the liver.

         • Lipoprotein Lipase (LPL): Synthesized in adipose tissue and muscle but secreted into the capillary endothelium to hydrolyze triglycerides in lipoproteins.

         • Pseudocholinesterase: Synthesized by the liver.

    2. Non-functional Plasma Enzymes ():**

       • Definition: These are enzymes whose site of action is intracellular. They have no known physiological function in the plasma.

       • Characteristics: Their concentration in plasma is normally very low or undetectable. Their presence reflects the normal, slow turnover of cells in the body.

       • Profound Clinical Significance: A significant increase in the plasma level of a non-functional, intracellular enzyme is a key indicator of cell damage or necrosis. When a cell's membrane is damaged, its intracellular contents, including enzymes, leak out into the interstitial fluid and then into the bloodstream.

       • The Principle: The magnitude of the enzyme elevation in plasma is roughly proportional to the extent of the tissue damage.

• B. Factors Affecting Plasma Enzyme Levels:

  • Rate of Release: The severity of tissue damage directly impacts how much enzyme leaks out.

  • Intracellular Location: Cytosolic enzymes appear in the plasma more readily than mitochondrial enzymes, which require more severe damage to be released.

  • Rate of Clearance: Enzymes are cleared from the plasma by the reticuloendothelial system at different rates. The half-life (t½) of an enzyme in the plasma determines how long its elevation will be detectable after an injury.

  • Tissue Specificity: The ideal diagnostic enzyme would be present in high concentration in only one specific tissue, so its elevation in plasma would unambiguously point to damage in that organ.

Part 2: Isoenzymes - The Key to Tissue Specificity (15 minutes)

• A. Definition of Isoenzymes (or Isozymes) ():**

  1. Isoenzymes are different molecular forms of an enzyme that catalyze the same chemical reaction.

  2. Properties: They differ in their:

     • Primary Structure: They have different amino acid sequences because they are coded by different genes or, more commonly, are assembled from different types of polypeptide subunits.

     • Physicochemical Properties: They have different electrophoretic mobilities, kinetic parameters (Km, Vmax), and stability to heat or chemical inhibitors.

  3. Structure: They are typically oligomeric (multi-subunit) proteins. The different combinations of subunits give rise to the different isoenzyme forms.

• B. Diagnostic Importance of Isoenzymes:

  1. The total activity of an enzyme (like CK or LDH) may be elevated in damage to several different organs. However, different tissues often synthesize and contain a unique pattern or preponderance of specific isoenzymes.

  2. Therefore, separating and quantifying the specific isoenzymes in the plasma provides much greater tissue specificity and diagnostic power, allowing the clinician to pinpoint the site of injury.

  3. Method of Separation: Primarily by electrophoresis, which separates them based on differences in their net electrical charge.

• C. Key Clinical Examples of Isoenzymes:

  1. Creatine Kinase (CK) / Creatine Phosphokinase (CPK):

     • Function: Catalyzes the reversible phosphorylation of creatine by ATP.

     • Structure: A dimer composed of two types of subunits: M (for Muscle) and B (for Brain). This gives rise to three isoenzymes.

     • The Three Isoenzymes:

       • CK-BB (CK1): Fastest moving on electrophoresis. Predominantly found in the brain and smooth muscle.

       • CK-MB (CK2): Intermediate mobility. The "cardiac isoenzyme." The heart contains about 40% CK-MB, while skeletal muscle contains <5%.

       • CK-MM (CK3): Slowest moving. The predominant isoenzyme in both skeletal muscle and cardiac muscle.

     • Diagnostic Use: A significant rise in total CK with a CK-MB fraction greater than 5-6% of the total is highly specific for acute myocardial infarction (MI).

  2. Lactate Dehydrogenase (LDH):

     • Function: Catalyzes the reversible conversion of lactate to pyruvate.

     • Structure: A tetramer composed of two types of subunits: H (for Heart) and M (for Muscle). This gives rise to five isoenzymes.

     • The Five Isoenzymes (LDH1 to LDH5):

       • LDH1 (H₄): Fastest. Predominant in heart muscle and RBCs.

       • LDH2 (H₃M₁): Predominant in heart and RBCs. (Normally, LDH2 > LDH1 in plasma).

       • LDH3 (H₂M₂): Found in lungs and other tissues.

       • LDH4 (H₁M₃): Found in liver and skeletal muscle.

       • LDH5 (M₄): Slowest. Predominant in liver and skeletal muscle.

     • 
       

     • Diagnostic Use:

       • In MI: LDH1 rises later than CK-MB. A characteristic "flipped pattern" where LDH1 > LDH2 is seen, which is highly suggestive of MI.

       • In Hepatitis: A marked elevation of LDH5 is seen.

       • In Hemolytic Anemia: Both LDH1 and LDH2 are elevated due to release from RBCs.

Part 3: Specific Enzymes as Diagnostic Markers (30 minutes)

• A. Enzymes in Myocardial Infarction ():**

  • Note: While enzymes were the historic gold standard, they have been largely superseded by the protein cardiac Troponins (cTnI and cTnT), which are more sensitive and specific. However, understanding the enzyme profile remains important.

  • Profile of Enzyme Release after MI:

    • Creatine Kinase (CK-MB): The first enzyme to appear in the blood. Starts to rise within 4-6 hours, peaks at ~24 hours, and returns to normal in 2-3 days. This provides a narrow diagnostic window.

    • Aspartate Aminotransferase (AST): Rises after CK-MB (~8-12 hours), peaks at 48 hours, returns to normal in 4-5 days. It is not specific as it is also high in liver disease.

    • Lactate Dehydrogenase (LDH1): The last to rise (~12-24 hours), peaks at 48-72 hours, and remains elevated for 7-10 days. It is useful for the late diagnosis of MI.

• B. Enzymes in Liver Disease ():**

  • Hepatocellular Damage (e.g., Viral Hepatitis, Toxic Injury):

    • Aminotransferases / Transaminases: These are the most sensitive markers of hepatocyte injury.

      • Alanine Aminotransferase (ALT): Primarily located in the cytosol of hepatocytes. It is considered more specific for liver damage.

      • Aspartate Aminotransferase (AST): Found in both the cytosol and mitochondria. Also present in high concentrations in heart and muscle.

    • Pattern: In most acute liver damage, ALT > AST. In alcoholic hepatitis, a characteristic pattern of AST > ALT (ratio > 2) is often seen, partly because alcohol causes mitochondrial damage, releasing mitochondrial AST.

  • Cholestasis (Obstructive Jaundice):

    • Alkaline Phosphatase (ALP): Found in the cells lining the bile canaliculi. When bile flow is obstructed, the synthesis of ALP is induced, and it regurgitates into the blood. Markedly elevated levels are a hallmark of cholestasis.

    • Gamma-Glutamyl Transferase (GGT): Also located on the biliary epithelium and is extremely sensitive to cholestasis. It is also induced by alcohol, making it a sensitive marker for alcohol-related liver disease.

• C. Enzymes in Pancreatic Disease ():**

  • Acute Pancreatitis: Inflammation and destruction of the acinar cells of the pancreas leads to the release of digestive enzymes into the circulation.

    • Serum Amylase: Rises rapidly within hours of onset, but is not very specific (also produced by salivary glands).

    • Serum Lipase: Rises slightly later than amylase but is much more specific for pancreatic damage and remains elevated for longer. It is the preferred marker for acute pancreatitis.

• D. Enzymes in Bone and Prostate Disease:

  • Bone Disease:

    • Alkaline Phosphatase (ALP): Produced by osteoblasts. Levels are elevated in conditions with increased osteoblastic activity, such as:

      • Physiological: During childhood growth, pregnancy.

      • Pathological: Paget's disease of bone, rickets, osteomalacia, healing fractures, and bone cancers (osteosarcoma).

  • Prostate Cancer:

    • Acid Phosphatase (ACP): High concentrations are found in the prostate gland. Elevated serum levels, particularly if they have spread beyond the prostate capsule, are indicative of metastatic prostate cancer.

    • Note: The protein Prostate-Specific Antigen (PSA) is a more sensitive and widely used marker for screening and monitoring prostate cancer today.