Enzyme Kinetics and Inhibition

Enzyme Kinetics

• Kinetics is the study of reaction rates (velocities).
• Studying enzyme kinetics is useful to measure:
  • Enzyme concentration in a mixture (by its catalytic activity)
  • Enzyme purity (specific activity)
  • Enzyme catalytic efficiency and/or specificity for different substrates
  • Comparison of different forms of the same enzyme in different tissues or organisms
  • Effects of inhibitors (revealing catalytic mechanism, active site structure, potential therapeutic agents)

Enzyme Kinetics Equation

• S = substrate
• E = enzyme
• P = product
• ES = enzyme-substrate complex
• k₁, k⁻₁, k₂, k⁻₂ are rate constants

Initial Velocity (v₀) and [S]

• Substrate concentration ([S]) greatly influences the rate of product formation (velocity, v).
• Studying the effect of [S] on v is complicated by the reversibility of enzyme reactions (e.g., conversion of product back to substrate).
• Initial velocity (v₀) measurements are used to overcome this problem.
• At the start of a reaction, [S] is much greater than [P], so the initial velocity depends on substrate concentration.
• Plotting v₀ against [S] results in a hyperbolic curve.
• At maximum velocity (Vmax), all the enzyme is saturated with bound substrate (meaning only the ES complex is present), if [S] >> E.

Substrate Saturation of an Enzyme

• Diagrams illustrating substrate saturation levels at low, 50%, and high [s].
• The diagrams show the interaction of substrate and enzyme at different substrate levels (low, 50%, saturating).

Michaelis-Menten Curve

• The Michaelis-Menten equation describes the hyperbolic relationship between v₀ and [S].
• At low [S], v₀ is directly proportional to [S].
• At high [S], v₀ approaches Vmax.
• Km is the Michaelis constant, where v₀ = ½ Vmax when [S] = Km.

Michaelis-Menten Equation

• The equation describes the dependence of velocity on substrate for many enzymes: v₀ = (Vmax[S]) / (Km + [S]).
• v₀ = initial reaction velocity
• Vmax = maximal velocity
• [S] = substrate concentration
• Km = Michaelis constant (can be graphically determined by measuring v₀ vs [substrate]).

Meaning of Km

• Km (the Michaelis constant) is a relative measure of the affinity of the enzyme for its substrate (identical to Kd).
• If v₀ = ½ Vmax, then [S] = Km.
• High Km = weak binding between enzyme and substrate (high dissociation); low Km = strong binding

Lineweaver-Burk (double reciprocal plot)

• The reciprocal of the Michaelis-Menten equation (1/v₀ = (Km/Vmax)(1/[S]) + 1/Vmax) yields a linear equation.
• Plotting 1/v₀ against 1/[S] results in a straight line.
• Slope = Km/Vmax
• Y-intercept = 1/Vmax
• Used to determine Vmax and Km accurately.
• Useful in characterizing enzyme inhibitors and different enzyme mechanisms.

Uses of Double Reciprocal Plot

• The x-intercept value is equal to -1/Km.
• The plot provides a more accurate determination of Vmax and Km.
• Useful for characterizing enzyme inhibitors and distinguishing between different enzyme mechanisms.

Enzyme Inhibitors

• Enzyme reaction rates, if lower than expected for pH, temperature, substrate, and activator concentrations, suggest inhibition.
• Inhibitors can be reversible (dissociate) or irreversible (permanently bind).

Reversible Inhibitors

• Competitive, non-competitive, uncompetitive inhibitors.
• Km is altered based on the inhibitor type (increased for competitive, no change for non-competitive, decreased for uncompetitive).
• Vmax is affected in competitive and non-competitive inhibition (not affected in competitive inhibition).
• Plots of v₀ against [S] show different effects depending on the inhibitor type.

Irreversible Inhibitors

• Result in the destruction or modification of essential amino acids.
• Often involve covalent interactions between the enzyme and inhibitor.
• Examples include: cyanide (cytochrome oxidase), heavy metals (combine with –SH groups), alkylating agents (e.g., iodoacetamide)

Specific Enzyme Inhibitors

• Regulate enzyme activity.
• Help understand enzyme action mechanisms. (Denaturing agents are not inhibitors.) Reversible inhibitors form an EI complex that dissociates back to enzyme and free inhibitor. Reversible inhibitors are classified based on their mechanism of action (competitive, non-competitive, and uncompetitive). Irreversible inhibitors form covalent or very tight permanent bonds with amino acids at the active site, rendering the enzyme inactive. (e.g. groups specific reagents, substrate analogs, suicide inhibitors).

Examples of Enzyme Inhibition

• Cyanide inhibits cytochrome oxidase by binding to its iron atom.
• Heavy metals (e.g., Ag⁺, Hg²⁺) combine with –SH groups on enzymes.
• Diisopropyl phosphofluoridate inhibits acetylcholinesterase (nerve gas example).
• Malonate is a reversible competitive inhibitor of succinate dehydrogenase. it resembles the substrate succinate, and binds to the active site, preventing succinate from binding.

This quiz covers fundamental concepts related to enzyme kinetics and various types of inhibition, including suicide inhibitors and competitive inhibition. Test your understanding of key terms and mechanisms that govern enzyme activity and reaction velocities. Ideal for students studying biochemistry or related fields.