Week 1

Enzyme Kinetics Overview

  • Enzyme kinetics investigates the rates of enzyme-catalyzed reactions and the factors affecting them.

  • Substrate specificity can cause a good substrate to be cleaved up to 10,000 times faster than a poor substrate.

Stages of Enzyme Reactions

  • Michaelis and Menten Model:

    • Reactions occur in two stages:

    1. Formation of the Enzyme-Substrate Complex:

      • The substrate (S) binds to the enzyme (E), forming an enzyme-substrate complex (ES).

      • This binding is quick and reversible, characterized by rate constants k₁ (binding) and k₂ (dissociation).

    2. Production of the Enzyme Product Complex:

      • Enzyme-bound substrate is converted to product (P), which then dissociates from the enzyme, with rate constants k₃ (formation of P) and k₄ (dissociation of P).

Critical Kinetic Variables

  • Two core variables define enzymatic performance:

    • Km (Michaelis Constant): A measure of the enzyme's affinity for its substrate.

    • Low Km indicates high affinity; high Km indicates low affinity.

    • Vmax (Maximal Velocity): The maximum rate of enzymatic reaction when the enzyme is saturated with substrate.

Key Concepts in Enzyme Kinetics

  • Reaction Rate vs. Reaction Velocity:

    • Velocity: Change in concentration of substrate or product per unit time.

    • Rate: Change in total quantity (moles, grams) per unit time.

Michaelis-Menten Equation

  • The effect of substrate concentration on reaction velocity is mathematically described by the Michaelis-Menten equation: V=V<em>max[S]K</em>m+[S]V = \frac{V<em>{max}[S]}{K</em>m + [S]}

    • Where:

      • V = Reaction velocity

      • [S] = Substrate concentration

      • Vmax = Maximal reaction velocity

      • Km = Substrate concentration at 1/2 Vmax

Assumptions of the Michaelis-Menten Model

  1. The substrate concentration [S] is large compared to the enzyme concentration [E], so the formation of the complex ES does not significantly change [S].

  2. At the beginning of the reaction, product [P] is negligible, allowing the reaction rate associated with k₄ to be ignored.

  3. The conversion of ES to E + P is rate-limiting (i.e., the slowest step in the reaction).

  4. The formation of the ES complex is rapid and reversible, maintaining a balance in formation and dissociation.

Reaction Velocity Behavior

  • At low substrate concentrations, velocity rises with [S].

  • As saturation occurs, increases in [S] yield diminishing returns in velocity up to Vmax.

  • Vmax indicates where the enzyme is fully saturated and cannot catalyze faster.

Influences on Km and Vmax

  • Both Km and Vmax are influenced by:

    • pH

    • Temperature

    • Ionic strength

    • Enzyme inhibitors

Double Reciprocal Plots (Lineweaver-Burk Plots)

  • To acquire linear data for estimating Vmax and Km:

    • The Michaelis-Menten equation is transformed:
      1V=K<em>mV</em>max1[S]+1Vmax\frac{1}{V} = \frac{K<em>m}{V</em>{max}} \frac{1}{[S]} + \frac{1}{V_{max}}

    • Slope = ( Km/V{max} )

    • y-intercept = ( 1/V_{max} )

    • x-intercept = ( -1/K_m )

Enzyme Inhibition

  • Enzyme action can be inhibited by chemicals, typically categorized into:

    • Irreversible Inhibitors: Bind tightly and do not dissociate quickly.

    • Example: Nerve gas diisopropyl-phosphofluoridate (DIPF) inhibits acetylcholinesterase (AChE).

    • Results in prolonged action of acetylcholine (ACh), potentially leading to toxicity.

    • Reversible Inhibitors: Can be competitive or noncompetitive.

Reversible Inhibitor Types

1. Competitive Inhibitors

  • Compete directly with substrate for active sites.

  • Sufficient substrate concentration can overcome inhibition.

  • Effects on kinetic parameters:

    • Vmax remains unchanged.

    • Apparent Km (Km') increases due to higher substrate needed to achieve the same reaction rate.

2. Noncompetitive Inhibitors

  • Bind to an allosteric site, different from active site, allowing for simultaneous binding of substrate and inhibitor.

  • Effects on kinetic parameters:

    • Km remains unchanged.

    • Vmax decreases (Vmax').

3. Uncompetitive Inhibitors

  • Bind only to the ES complex, affecting both Km and Vmax.

  • Important in reactions with two substrates.

4. Irreversible Inhibitors

  • Form stable, permanent complexes with enzymes, permanently inactivating them.

  • Influence on kinetics similar to noncompetitive inhibition (Vmax decreases, Km unaffected).

Isozymes

  • Isozymes (or isoenzymes) are enzyme variants that catalyze similar reactions but differ slightly in structure and kinetics.

  • Often organ-specific, making them useful for diagnostics.

  • Key examples include:

    • Creatine Phosphokinase (CPK) and Lactate Dehydrogenase (LDH).

Diagnostic Utility of Isozymes

  • Elevated specific isozymes in plasma reflect cellular damage or pathological conditions.

  • Each isozyme typically has a distinct clinical significance reflected in diagnostic tests.

Cofactors and Coenzymes

  • Enzymatic activity often relies on nonprotein molecules known as cofactors.

    • Holoenzyme: Enzyme with its cofactor.

    • Apoenzyme: Enzyme without its cofactor, often inactive.

  • Types of cofactors include:

    • Metal Ions: (e.g., Zn²⁺, Mg²⁺, Mn²⁺)

    • Coenzymes: Organic molecules that act as carriers of chemical groups or electrons during reactions.

    • Examples of coenzymes derived from vitamins include NAD⁺, FAD, and Coenzyme A.

Objectives of Study

  • Define Km and Vmax of enzyme-catalyzed reactions, exploring their determinants.

  • Discuss the relationship of Km with enzyme affinity.

  • Compare effects of competitive vs noncompetitive inhibitors on kinetic variables.

  • Examining the use of isozymes for diagnosis, and understanding the role of cofactors and coenzymes in enzymatic reactions.

Review Questions

  1. The variable that measures the affinity of an enzyme for its substrate is:

    • b. Km

  2. Temperature and pH affect:

    • e. Km and Vmax

  3. The maximal velocity of an enzyme-catalyzed reaction is:

    • e. Decreased in the presence of an irreversible inhibitor.

  4. Tissue with the highest concentration of MB dimer for creatine phosphokinase is:

    • c. Heart

  5. A therapeutic inhibitor considered to be a "suicide substrate" is:

    • a. Aspirin

  6. The y-intercept on a lineweaver-Burk plot is:

    • c. 1/Vmax

  7. Correct representation of the Michaelis-Menten equation is:

    • c. V=V<em>max[S]K</em>m+[S]V = \frac{V<em>{max}[S]}{K</em>m + [S]}

  8. All of the following are TRUE of competitive inhibitors, EXCEPT:

    • b. They reduce the apparent Km (Km') of the reactions they inhibit.

  9. An enzyme inhibitor affecting both Vmax and Km is:

    • b. Uncompetitive