Enzymes Notes

Introduction

• In living cells, hundreds of biochemical reactions occur rapidly and simultaneously.
• Enzymes are responsible for the orderliness and speed of these reactions at modest temperatures.

Roles of Enzymes in Cells

• Enzymes have two fundamental roles:
  • Catalysis: They act as highly specific biological catalysts, accelerating metabolic reactions by breaking down or assimilating substances.
  • Regulation: They regulate chemical reactions; their quantity and activity determine reaction rates.

Nature of Enzymes

• Most enzymes are globular proteins, comprising one or more polypeptides coiled and folded into a globular structure.
• Some enzymes are made of RNA (ribozymes).
• Enzymes can be simple or conjugated with a cofactor (non-protein prosthetic group).
• Their action depends on their specific 3D conformation.
• Enzymes are organic catalysts produced by living cells, remaining unchanged at the reaction's end and not altering the end products' nature or properties.
• Enzymes alter reaction rates but not chemical equilibrium.
• Enzymes are efficient; small amounts can catalyze many reactions.
  • Example: Catalase can break down 600,000 moles of H2O2 in 1 second at 37°C.
• Enzymatic reaction rates are affected by pH, temperature, enzyme concentration, and substrate concentration.
• Enzymes are highly specific due to their specific 3D conformation, where the active site complements the substrate.

Enzyme Specificity

• Enzymes can exhibit:
  • Absolute specificity: Catalyzing a single, specific reaction.
    • Example 1: Sucrase catalyzes only the breakdown of sucrose into glucose and fructose: Sucrose \xrightarrow{sucrase} glucose + fructose
    • Example 2: Catalase catalyzes only the decomposition of hydrogen peroxide: 2H2O2 \xrightarrow{catalase} 2H2O + O2
  • Group specificity: Attacking a type of chemical bond.
    • Example: Trypsin digests the peptide bond at the carboxyl side of lysine or arginine in any polypeptide.
• Enzymes are named by adding the suffix -ase to the substrate name (e.g., amylase hydrolyses amylose).
  • Exceptions exist, like catalase, trypsin, and renin.

Enzyme Structure

• Enzymes may consist of one or more polypeptide chains, each folding into secondary and tertiary structures.
• The enzyme has an active site, which is the catalytic center where the substrate binds for catalysis.
• Some amino acid residues form the active site, while others serve a structural role.

Common Features of Active Sites

1. The active site is a three-dimensional cleft formed by R groups of amino acids.
   • It has a specific 3D conformation complementary to the substrate, accounting for substrate specificity.
2. The active site occupies a small part of the total enzyme volume.
   • Typically consists of 3-12 amino acids brought together through folding.
3. Substrates bind to enzymes at the active site via weak interactions such as ionic and hydrogen bonds.
4. Specificity of binding depends on the arrangement of amino acids in the active site.

Types of Amino Acid Residues in Enzymes

1. Binding residues: Found at the active site and bind to the substrate.
   • May consist of amino acids with charged acidic or basic R groups that form ionic bonds with the substrate.
   • May consist of amino acids with polar R groups that form hydrogen bonds with the substrate.
2. Catalytic residues: Found at the active site and catalyze the reaction.
3. Structural residues: Form the support structure for the active site.
4. Non-essential residues: Have no specific role.

Mechanism of Enzyme Action

• Substrate molecules must gain a certain amount of energy, called activation energy (E_a), before chemical changes are possible.
• A chemical reaction of substrate (S) to form product (P) goes through a transition state that has a higher free energy than either (S) or (P).
• The transition state is the most short-lived state along the reaction pathway because it is the most unstable and therefore has the highest free energy.
• Substrate must gain energy from their surroundings to form the transition state.

Overcoming the Activation Energy Barrier

The activation energy barrier can be overcome by:

• Increasing the free energy of the substrate (e.g., by increasing temperature and pressure) to speed up the movements of reacting molecules.
• The addition of an enzyme/catalyst. Enzymes lower the activation energy required to form the transition state at a moderate temperature. *Note: Free energy is the energy from a system that is available to do work.
  • Enzymes help to form a transition state that is more stable using:
    • Proximity effect: Temporary binding of substrates next to each other on an enzyme increases the chance of a reaction. Uncatalyzed reactions depend on random collision between substrate molecules.
    • Strain effect: Slight distortion of the substrates as they bind to the enzyme causes bonds of the substrate to be strained, thereby increases the chance of breakage.
    • Orientation surface: The substrate may be held in such a way that exposes it for reaction.
    • Microenvironment effect: Hydrophobic amino acids create a water-free zone in which non-polar substrates may react more easily.
    • Acid–base catalysis: Acidic and basic amino acids in the enzyme facilitate the donation of protons to or acceptance of protons from the substrate.

Models of Enzyme Action

• Lock-and-key hypothesis (Fisher, 1894): The enzyme and substrate are exactly complementary. The enzyme (E) and substrate (S) molecules collide in the correct orientation, forming a short-lived enzyme-substrate (ES) complex. Once the reaction has occurred, the product and free enzyme are released from the former ES complex.
• Induced-fit model (Koshland, 1959): When the substrate binds with the active site of an enzyme, it induces a 3D conformational change in the enzyme structure. The enzyme ‘wraps around’ the substrate to form a more stable structure.

Enzyme Cofactors

• Many enzymes require non-protein components called cofactors for efficient activity.
• Cofactors are required for enzyme activity but they are not catalytic on their own.
• There are 3 main types of cofactors:
  • Inorganic ions: Can be tightly or loosely associated with an enzyme, molding it into a conformation for easy formation of the ES complex.
    • Example 1: Cl^- for salivary amylase
    • Example 2: Ca^{2+} for phosphorylase kinase
  • Coenzymes: Non-protein, organic cofactors loosely associated with an enzyme and derived from vitamins.
    • Example: NAD^+ (Nicotinamide Adenine Dinucleotide) for dehydrogenase. It functions as a hydrogen acceptor.
  • Prosthetic Groups: Non-protein, organic cofactors tightly bound to the enzyme permanently.
    • Example: Iron-containing heme for hemoglobin.

Rate of Enzyme-Catalyzed Reactions

• The rate can be determined by measuring product formation or substrate disappearance over time.
• The choice of method depends on the convenience of sampling.
  • Product formation (e.g., measuring O2 produced in H2O_2 decomposition).
  • Substrate disappearance (e.g., measuring starch disappearance using iodine in starch hydrolysis by amylase).

Factors Affecting Rate of Enzyme-Catalyzed Reactions

The 4 major factors affecting the rate of enzyme reaction are:

1. enzyme concentration,
2. substrate concentration,
3. temperature and
4. pH.

• In addition, the presence of inhibitors (both competitive and non-competitive) also affects the rate of enzyme reaction.
• When investigating the effect of a given factor on the rate of an enzyme reaction, all other factors should be kept constant and at optimum levels.

Effect of Enzyme Concentration

• When substrate concentration is maintained at a high level, and other conditions such as temperature and pH are kept constant, the rate of reaction is proportional to the enzyme concentration.
• As enzyme concentration increases, more active sites are available for substrate binding, increasing successful collisions and the rate of enzyme-substrate complex formation.

Effect of Substrate Concentration

• For a given enzyme concentration, the reaction rate increases with increasing substrate concentration until V_{max} is reached.
• At low substrate concentrations, available enzymes have unoccupied active sites.
• At high concentrations, active sites are saturated, and extra substrate must wait for ES complex dissociation.
• The Michaelis constant (Km) is the substrate concentration at half of V{max}, indicating enzyme-substrate affinity.
  • Low K_m: High affinity.
  • High K_m: Low affinity.

Effect of Temperature

• At very low temperatures, enzymes are inactivated due to decreased kinetic energy and collision frequency.
• Increasing temperature increases kinetic energy, ES complex formation, and product formation.
• The temperature coefficient (Q{10}) expresses the effect of temperature on reaction rate: Q{10} = \frac{Rate \space of \space reaction \space at \space x + 10\degree C}{Rate \space of \space reaction \space at \space x \degree C}
• An optimum temperature is soon reached, corresponding to the maximum rate of reaction.
• Most enzymes have optimum temperatures in the range of 35 – 40°C, but there are exceptions, such as thermophilic bacterial enzymes, used in biological washing powders where the optimum temperature can be 70°C.
• Above the optimum temperature, the reaction rate falls off sharply.
• High temperatures denature enzymes by breaking weak attractive forces, altering the 3D conformation of the active site.
• Low temperature inactivates enzymes reversibly.

Effect of pH

• Enzymes function within a narrow pH range; activity decreases outside this range.
• Optimum pH is the pH at which the maximum rate of reaction occurs.
• pH affects enzyme activity in two ways:
  • Ionization of binding and catalytic residues at the active site: Changes in pH alter the ionic charges of amino acid residues, inhibiting binding or catalysis.
  • Change in 3D conformation of the active site: pH changes disrupt ionic and hydrogen bonding, altering the active site's conformation and decreasing enzyme activity. At extreme pH, the enzyme is denatured.

Enzyme Inhibition

• Enzyme inhibitors reduce the rate of enzyme-controlled reactions.
  • Many medicinal drugs and poisons function as enzyme inhibitors.
• Inhibition can be reversible or irreversible.

Reversible vs. Irreversible Inhibition

*   **Reversible Inhibition**: The inhibitor does not permanently affect the enzyme and can resume its function when the inhibitor is removed. It binds through weak interactions (e.g., hydrogen bonds).
    *    Example: Cyanide is a reversible inhibitor of enzyme cytochrome oxidase.
*   **Irreversible Inhibition**: These inhibitors bind to enzymes permanently, making it difficult to restore the enzyme activity. These inhibitors cause the enzyme proteins to precipitate. The irreversible inhibitor binds with the enzyme through strong bonds (e.g. covalent bonds)
    *   Example 1: Heavy metal ions, e.g., mercury, arsenic and silver
    *   Example 2: Nerve gas DFP (diisopropylfluorophosphate)

Competitive vs. Non-competitive Inhibition

*   **Competitive Inhibition**: The inhibitor has a structural resemblance to the substrate molecule and is complementary to the specific 3D conformation of the active site, therefore, competing for binding at the same active site.
    *   Example: Malonate competes with succinate for succinate dehydrogenase.
*   **Non-competitive Inhibition**: The inhibitor forms an enzyme-inhibitor complex by binding to a site on the enzyme other than the active site (also known as the allosteric site for allosteric enzymes).
    *   The binding of the non-competitive inhibitor causes the enzyme to change its 3D conformation.
    *   Increasing substrate concentration has no effect on the rate of reaction.

Allosteric Inhibition

• Allosteric enzymes are multi-subunit enzymes with an allosteric site distinct from the active site.
• Molecules binding to the allosteric site act as allosteric effectors (activators or inhibitors).
• The enzyme exists in active and inactive states depending on effector binding.

Compared Non-competitive inhibition and Allosteric regulation

Roles of Enzymes in Metabolic Regulation and Control

• Cells control enzymatic reactions through:
  1. Membrane compartmentalization: Physical separation of substrates and enzymes.
  2. Variation in pH: Alters the rate of enzyme reactions.
  3. Controlling substrate concentration.
  4. Using regulatory enzymes and feedback control.

Feedback Control

• Resources in a plant or animal body are usually limited. Hence, enzymes or products should only be produced when necessary.
• Feedback control regulates active enzyme concentration.
  • Negative Feedback: Output inhibits the system.
  • End-product inhibition is a Negative Feedback control example.
• Regulatory enzymes are within metabolic pathways.
  1. In a linear metabolic pathway, enzymes are arranged in order
  2. Each enzyme is inter-dependent upon the ones adjacent to it
  3. When the end-product of a metabolic pathway begins to accumulate, it may act as an inhibitor on the enzyme controlling the first step of the pathway.
  4. The affinity of the enzyme for its substrate is therefore lowered, and further production of the end-product is decreased or prevented.
  5. This linear order of the enzymes permits self-regulation by negative feedback inhibition, whereby the rate of the metabolic pathway is controlled by the concentration of the end-product.

Example of negative feedback

• ATP on enzyme Phosphofructokinase (PFK)
  • A high concentration of ATP inhibits the enzyme PFK.