Enzymes: Biological Catalysts

Key Vocabulary

• Catalyst: A substance that increases the rate of a chemical reaction without being consumed.

• Enzyme: A biological macromolecule (usually a protein) that acts as a highly specific catalyst in biochemical reactions.

• Active site: The region on an enzyme where substrate molecules bind and undergo a chemical reaction.

• Substrate: The reactant molecule upon which an enzyme acts.

• Coenzyme: An organic non-protein molecule that binds to the enzyme and is required for its activity.

• Cofactor: A non-protein chemical compound (can be inorganic or organic) required for enzyme activity.

• Prosthetic group: A tightly or covalently bound cofactor or coenzyme essential for enzyme function.

• Allosteric: Refers to regulation of an enzyme by binding an effector molecule at a site other than the active site.

• Rate constant (k): A proportionality constant in the rate equation of a reaction.

• Half-life (t1/2): The time required for half of the reactant to be converted to product.

• Rate-limiting: The slowest step in a metabolic pathway, determining the overall rate.

• Standard free energy of activation (ΔG°‡): The energy barrier that must be overcome for a reaction to proceed.

• Transition state: A high-energy, unstable state during a reaction, representing the point of highest energy along the reaction path.

• Reaction rate/velocity (v): The speed at which substrate is converted to product.

• Rate enhancement: The increase in reaction rate due to the presence of a catalyst.

• Ideal conditions: Experimental conditions under which enzyme activity is measured (e.g., optimal pH, temperature, substrate concentration).

• Initial velocity (v0): The rate of reaction when the substrate concentration is high and product formation is linear with time.

• Vmax: The maximum velocity of an enzyme-catalyzed reaction when the enzyme is saturated with substrate.

• kcat: The turnover number; the number of substrate molecules converted to product per enzyme molecule per unit time at saturation.

• KM: The Michaelis constant; the substrate concentration at which the reaction velocity is half of Vmax.

• Saturation: The state when all enzyme active sites are occupied by substrate.

• Affinity: The strength of the interaction between enzyme and substrate; lower KM indicates higher affinity.

• Isozyme/isoenzyme: Different molecular forms of an enzyme that catalyze the same reaction but differ in structure or regulation.

• X- and Y-intercept: Intercepts on a Lineweaver-Burk plot used to determine kinetic parameters.

• Double reciprocal: Refers to the Lineweaver-Burk plot, a double reciprocal plot of 1/v versus 1/[S].

• Reversible/irreversible inhibition: Types of enzyme inhibition; reversible can be undone, irreversible cannot.

• Competitive, Noncompetitive, Uncompetitive inhibition: Types of reversible inhibition based on inhibitor binding site and effect on kinetics.

• Activation: Increase in enzyme activity by various mechanisms.

• Effector molecules: Molecules that modulate enzyme activity (activators or inhibitors).

• NSAID: Non-steroidal anti-inflammatory drug; inhibits enzymes involved in inflammation.

• COX: Cyclooxygenase; an enzyme targeted by NSAIDs.

• Feedback inhibition: A regulatory mechanism where the end product of a pathway inhibits an upstream step.

• Homeostasis: Maintenance of stable internal conditions.

• Phosphorylation/kinase: Addition of a phosphate group to a molecule by a kinase enzyme.

• Dephosphorylation/phosphatase: Removal of a phosphate group by a phosphatase enzyme.

• Zymogen: An inactive enzyme precursor that requires modification to become active.

• Proprotein: An inactive precursor of a protein, requiring processing for activation.

• Ribozyme: An RNA molecule with catalytic activity.

Cofactors, Coenzymes, and Prosthetic Groups

Definitions and Differences

• Cofactor: A non-protein chemical compound required for enzyme activity. Can be inorganic (e.g., metal ions like Mg2+, Zn2+).

• Coenzyme: An organic molecule (often derived from vitamins) that temporarily binds to the enzyme and participates in the reaction (e.g., NAD+, FAD).

• Prosthetic group: A cofactor or coenzyme that is tightly or covalently bound to the enzyme (e.g., heme in hemoglobin).

Enzyme-Substrate Interaction Models

Lock-and-Key vs. Induced Fit

• Lock-and-Key Model: The enzyme's active site is a rigid structure complementary to the substrate's shape.

• Induced Fit Model: The enzyme's active site is flexible and molds itself around the substrate upon binding, enhancing specificity and catalysis.

• Favored Model: The induced fit model is favored because it explains the dynamic nature of enzyme-substrate interactions and the stabilization of the transition state.

Reaction Order and Kinetics

First-Order vs. Second-Order Reactions

• First-order reaction: Rate depends linearly on the concentration of one reactant. Units of rate constant (k1): s-1.

• Second-order reaction: Rate depends on the product of the concentrations of two reactants. Units of rate constant: M-1s-1.

• Order Determination: Determined experimentally by measuring how the rate changes with varying reactant concentrations.

• v (nu): Symbol for reaction velocity; units are typically M/s (moles per liter per second).

• Enzyme reactions are theoretically second-order (depend on both enzyme and substrate), but under saturating substrate, they become pseudo-first-order.

• Half-life (t1/2): For a first-order reaction,

Temperature and Reaction Rate

Effect of Temperature

• Increasing temperature generally increases reaction rate by providing more energy to overcome the activation barrier.

• However, biological systems cannot simply increase temperature; instead, they use enzymes to lower the activation energy.

• Enzymes allow reactions to proceed rapidly at physiological temperatures.

Transition State and Activation Energy

Enzyme Mechanism

• Enzymes stabilize the transition state, lowering the activation energy (ΔG°‡) required for the reaction.

• Although the transition state is energetically unfavorable, enzymes make the reaction favorable by stabilizing intermediates through noncovalent interactions (hydrogen bonds, van der Waals forces, ionic interactions).

• Enzymes do not change the overall free energy change (ΔG°) of the reaction; they only lower the activation energy.

Rate Enhancement and Catalytic Mechanisms

How Enzymes Accelerate Reactions

• Rate enhancement: Enzymes can increase reaction rates by factors of 106 to 1012 compared to uncatalyzed reactions.

• Mechanisms include:

  • Proximity and orientation effects

  • Transition state stabilization

  • Acid-base catalysis

  • Covalent catalysis

  • Metal ion catalysis

• Enzymes often use multiple mechanisms simultaneously (e.g., lysozyme and chymotrypsin).

Active Site of Lysozyme

Key Residues and pH Dependence

• Lysozyme's active site contains Glu35 (glutamic acid at position 35) and Asp52 (aspartic acid at position 52).

• These residues are crucial for catalysis: Glu35 acts as a general acid, Asp52 stabilizes the transition state.

• Lysozyme is most active between pH 4 and 6, where the ionization states of Glu35 and Asp52 are optimal for catalysis.

Michaelis-Menten Kinetics

Steady-State Assumption and Experimental Conditions

• The steady-state assumption states that the concentration of the enzyme-substrate complex remains constant during the initial phase of the reaction.

• Experiments are conducted under initial velocity conditions, with substrate in excess and product formation measured before significant substrate depletion or product inhibition occurs.

Michaelis-Menten Equation and Parameters

• The Michaelis-Menten equation:

• KM (Michaelis constant): Indicates the substrate concentration at which the reaction rate is half-maximal. Lower KM means higher affinity.

• Vmax: Maximum velocity when the enzyme is saturated with substrate.

• kcat: Turnover number;

• kcat/KM: A measure of catalytic efficiency.

Graphical Analysis

• Michaelis-Menten plot: v vs. [S]; hyperbolic curve.

• Lineweaver-Burk plot: Double reciprocal plot (1/v vs. 1/[S]); linearizes the data.

• X-intercept:

• Y-intercept:

Enzyme Inhibition

Types of Reversible Inhibition

• Irreversible inhibition: Inhibitor covalently binds to the enzyme, permanently inactivating it.

Allosteric Regulation and Feedback Control

Mechanisms of Regulation

• Allostery: Regulation of enzyme activity by binding of effector molecules at sites other than the active site, causing conformational changes.

• Substrate-level control: Regulation by substrate concentration.

• Feedback inhibition: End product of a pathway inhibits an early enzyme, maintaining homeostasis.

NSAIDs and Enzyme Inhibition

Mechanism and Comparison

• NSAIDs (Non-steroidal anti-inflammatory drugs) inhibit COX (cyclooxygenase) enzymes, blocking prostaglandin synthesis and reducing pain and inflammation.

• Aspirin: Irreversible inhibitor; acetylates a serine residue in COX.

• Ibuprofen: Reversible competitive inhibitor; binds non-covalently to COX.

Reversible Covalent Modification

Phosphorylation and Dephosphorylation

• Phosphorylation (by kinases) and dephosphorylation (by phosphatases) are common reversible modifications regulating enzyme activity.

• Reversibility allows dynamic regulation in response to cellular signals.

Zymogens and Proproteins

Activation and Irreversibility

• Zymogen: Inactive enzyme precursor activated by irreversible covalent modification (e.g., cleavage of peptide bonds).

• Example: Trypsinogen (zymogen) is converted to trypsin (active enzyme) in the digestive tract.

• Irreversible activation prevents premature enzyme activity.

Ribozymes

RNA Catalysts

• Ribozyme: RNA molecule with catalytic activity.

• Example: The peptidyl transferase activity of the ribosome (involved in protein synthesis) is catalyzed by rRNA, a ribozyme.

Additional info: Some explanations and examples were expanded for clarity and completeness, such as the detailed mechanisms of inhibition, the Michaelis-Menten equation, and the role of lysozyme active site residues.