Lecture_07_Enzyme_Kinetics

Enzyme Kinetics Overview

• Enzyme kinetics studies the rates of biochemical reactions catalyzed by enzymes.

• Mechanisms by which enzymes increase reaction rates are fundamental to understanding their functions.

Enzyme-Catalyzed Reaction Pathway

• Enzyme (E) binds to its substrate (S) to form an enzyme-substrate complex (ES).

• A biochemical reaction occurs yielding a product (P), and the enzyme is regenerated to catalyze again.

• Significant transition states and enzyme-product complexes exist but are simplified here for basic understanding.

Rate Constants in Enzyme Reactions

• Rate constants for various steps in the reaction:

  • Formation of ES (E + S → ES): k1

  • Reversion (ES → E + S): k-1

  • Breakdown of ES to produce P (ES → E + P): k2

  • Back reaction (E + P → ES): k-2

• Rates of reaction are fastest for k1 and k-1 (larger constants), and slower for k2 and k-2 (smaller constants).

Initial Rates of Reaction

• Reaction rates increase with higher initial substrate concentrations.

• V0 denotes the initial rate of enzyme reaction for varying substrate levels as shown by dashed lines on plots.

• Experimental measures aim to capture V0 across different substrate concentrations.

Michaelis-Menten Equation

• The Michaelis-Menten equation relates reaction velocity (V) to substrate concentration (S):

  • V = (Vmax * S) / (S + Km)

  • V = observable reaction velocity (product production rate)

  • Vmax = maximum rate achievable under specific conditions

  • S = substrate concentration

  • Km = Michaelis constant indicating substrate concentration at half-maximal velocity.

• Plots of V0 vs S create a hyperbolic curve characteristic of enzyme kinetics.

Behavior of the Michaelis-Menten Plot

• At low S, the relationship is linear (V = K[S]), indicating proportional reaction velocity.

• At intermediate concentrations, a curve indicates the need for the full M-M equation.

• At S = Km, V = ½ Vmax highlighting enzyme efficiency.

• High S leads to saturation where additional substrate does not increase reaction rate, approximating Vmax.

Significance of Km

• Km is affected by three kinetic constants (k1, k-1, k2) and is a measure of enzyme efficiency:

  • Low Km indicates high efficiency at low substrate levels.

  • Km values range widely, indicating various enzyme efficacies (e.g. 10^-7 M to 10^-1 M).

Turnover Number (Kcat)

• Kcat = Vmax / [Et]

• Represents the number of substrate molecules converted to product per second per enzyme molecule.

• Kcat values illustrate efficiency; ranges from 0.5 to 40,000,000.

The Lineweaver-Burk Plot

• L-B plot is obtained by inverting the M-M equation, facilitating analysis of kinetic data.

  • Formula: 1/V = 1/Vmax + (Km/Vmax)(1/S).

• Straight-line graphs allow for easy extrapolation of data indicating Vmax and Km.

Enzyme Inhibition Overview

Types of Inhibitors

• Inhibitors can be reversible or irreversible, affecting enzyme activity.

• Reversible inhibitors can be overcome by removing the inhibitor or increasing substrate concentration.

  • Competitive inhibitors: Compete with substrate for active site occupancy.

  • Non-competitive inhibitors: Bind at sites other than the active site, inducing conformational changes.

• Irreversible inhibitors: Permanently modify enzymes, preventing activity.

Effects of Competitive Inhibition

• Competitive inhibitors increase apparent Km but do not affect Vmax.

• The relative concentration of substrate and inhibitor determines enzyme activity.

• Example: Methotrexate as a competitive inhibitor in cancer treatment, mimicking dihydrofolate.

Effects of Non-Competitive Inhibition

• Non-competitive inhibition does not change Km, but decreases Vmax.

• Substrates can still bind but do not produce product in the presence of non-competitive inhibitors.

Review of Kinetics

Irreversible Inhibitors

• Covalently modify proteins and often target active site residues.

• Examples include TPCK, which reacts with chymotrypsin, and DIFP, which modifies serine residues in serine proteases.

Conclusion

• Understanding enzyme kinetics and inhibition is paramount in biochemistry, affecting both research and therapeutic approaches.