Biochemistry Chapter: Enzyme Kinetics

Enzyme Kinetics and Calculations

• Biochemistry studies the chemical processes within and relating to living organisms. Enzyme kinetics explores how enzymes catalyze biochemical reactions.

Chemical Kinetics: The Law of Mass Action

• Reaction rate is often directly proportional to reactant concentrations.
• For the reaction A → B, the reaction rate (v₀) is proportional to [A].

Effect of [A] on Initial Rate

• A linear relationship often exists between initial reaction rate (v₀) and reactant concentration ([A]).
• This implies a first-order reaction.

Enzyme Kinetics: Importance of Enzymes

• Life processes rely on proteins, with cells containing thousands of diverse enzymes for myriad reactions.
• Approximately 20,000 protein genes in the human genome are related to enzymes.

Enzymes as Biological Catalysts

• Enzymes significantly accelerate chemical reactions compared to uncatalyzed reactions.
• Enzyme speed enhancements range from 10³ to 10⁶ times faster.

Rate Enhancement by Some Enzymes

• Different enzymes vary greatly in their rate enhancements.
• Table provided shows examples like OMP Decarboxylase (1.4 x 10¹⁷), AMP nucleosidase (6.0 x 10¹²), and others.

Enzyme Mechanism

• Induced Fit: Substrates bind to the active site, inducing the enzyme to change shape, enfolding the substrate.
• Substrates are held in the active site via weak interactions (hydrogen bonds, ionic bonds)
• Active sites can lower the activation energy, speeding up the reaction.
• Products are subsequently released.

Michaelis-Menten Kinetics

• Michaelis and Menten proposed a model to describe substrate saturation in enzyme-catalyzed reactions.
• The model involves substrate (S) binding to enzyme (E) to form an enzyme-substrate complex (ES) which then breaks down into enzyme (E) and product (P).

Effect of Substrate Concentration on Rate

• At low substrate concentrations ([S]), the initial reaction velocity (v₀) is approximately proportional to [S].
• At high substrate concentrations ([S]), v₀ becomes independent of [S] due to enzyme saturation.

Michaelis-Menten Equation

• V₀ = (V_max*[S]) / (K_m + [S])
• V₀ : Initial velocity of a reaction
• V_max : Maximum velocity of the reaction
• K_m : Michaelis constant, [S] required for the reaction to proceed at V_max/2.
• [S] : Substrate concentration

Meaning of K_m and V_max

• K_m indicates the substrate dependency (typically 10^-1 - 10^-6M).
• V_max represents the maximum rate of reaction. This is a measure of catalytic activity.

Units of Enzyme Activity

• Standard units: International Unit (IU, µmol/min)
• (SI unit): Katal (mol/sec) – a very large unit in practical terms.

Determining K_m and V_max

• Methods such as colorimetric assays are used for experimental determination of K_m and V_max.

Michaelis-Menten Plot & Problems

• Plots v₀ against [S] produce a hyperbolic curve approaching V_max at high [S].
• Determining exact values for V_max and K_m is challenging from direct plots

Lineweaver-Burk Plot

• Transforming the Michaelis-Menten equation gives: 1/V₀ = (K_m/V_max) * (1/[S]) + 1/V_max
• Plotting 1/V₀ against 1/[S] results in a straight line from which K_m and V_max can be determined.

Reaction Progress Curve

• Enzyme-catalyzed reactions show an initial phase of rapid increase in velocity that gradually plateaus.

Factors Affecting Reaction Rate

• Substrate availability decreases
• Product buildup: Reverse reaction becomes important.
• Enzyme degradation occurs.

Other Calculations for Kinetics

• Turnover Number (kcat): Represents the maximum catalytic activity of enzymes.
• The equation: V_max/([Enzyme_total]).

Enzyme Characteristics: Summary

• K_m and V_max are key parameters defining an enzyme's behavior.
• K_m reflects substrate affinity while V_max represents overall efficiency.