1. Maximum Velocity (Vmax)

• Definition: Vmax is the maximum rate at which an enzyme-catalyzed reaction can proceed when the enzyme is fully saturated with its substrate. At this point, all enzyme active sites are continuously occupied, and increasing the substrate concentration further will not increase the reaction rate.
• Units: Typically expressed as concentration per unit time (e.g., µM/min, mM/s).
• Interpretation:
  • Capacity: Vmax reflects the maximum catalytic capacity of a given amount of enzyme. It tells you the "top speed" of the enzymatic reaction under ideal, saturating conditions.
  • Enzyme Concentration Dependent: Vmax is directly proportional to the total enzyme concentration ([E]total). If you double the enzyme concentration, you double Vmax.
  • Limiting Step: It is determined by the rate-limiting step of the catalytic cycle when the enzyme is saturated.

2. Michaelis Constant (Km)

• Definition: Km is the substrate concentration at which the reaction velocity (V0) is exactly half of Vmax.
  • V0 = Vmax / 2 when [S] = Km.
• Units: Expressed in concentration units (e.g., M, mM, µM).
• Interpretation:
  • Substrate Affinity: Km is often interpreted as an inverse measure of the enzyme's apparent affinity for its substrate.
    • A low Km indicates that the enzyme reaches half Vmax at a low substrate concentration, meaning it has a high affinity for that substrate and can work efficiently even when substrate is scarce.
    • A high Km suggests a low affinity, requiring a higher substrate concentration to achieve half Vmax.
  • Physiological Relevance: It provides insight into the typical intracellular concentration range of the substrate where the enzyme is most sensitive to changes in substrate levels.
  • Not a Dissociation Constant (Kd): While Km can approximate the dissociation constant (Kd) of the enzyme-substrate (ES) complex (if Kcat is much smaller than the dissociation rate of ES), it is fundamentally a composite constant reflecting rates of substrate binding, dissociation, and product formation.

3. Turnover Number (Kcat)

• Definition: Kcat is a first-order rate constant that represents the maximum number of substrate molecules converted to product per enzyme active site per unit time when the enzyme is fully saturated with substrate.
• Units: Typically expressed in s⁻¹ (per second), often referred to as "per second" (e.g., 100 s⁻¹ means one enzyme molecule converts 100 substrate molecules per second).
• Calculation:
  Kcat = Vmax / [E]total
  Where [E]total is the total enzyme concentration.
• Interpretation:
  • Intrinsic Catalytic Rate: Kcat is a direct measure of the intrinsic catalytic efficiency of a single enzyme molecule. It tells you "how fast the enzyme can actually work" once it has bound the substrate and is operating at full capacity.
  • Enzyme Property: Unlike Vmax, Kcat is an intrinsic property of the enzyme for a specific substrate under defined conditions, independent of the total enzyme concentration used in the experiment.
  • Rate-Limiting Step: A high Kcat indicates a highly efficient enzyme that rapidly converts substrate to product. It directly reflects the rate-limiting step of the catalytic cycle under saturating conditions.

4. Catalytic Efficiency (Kcat/Km / Specificity Constant)

• Definition: The ratio of the Turnover Number (Kcat) to the Michaelis Constant (Km). It is a second-order rate constant.
• Units: M⁻¹s⁻¹ (or similar concentration⁻¹time⁻¹ units).
• Interpretation:
  • Overall Enzyme Effectiveness: This ratio is considered the best measure of an enzyme's overall catalytic efficiency, particularly at low (physiological) substrate concentrations where the enzyme is not saturated. It reflects how well the enzyme can bind substrate and convert it to product.
  • Specificity: It is also known as the specificity constant because it allows for the comparison of an enzyme's preference for different competing substrates. An enzyme will preferentially act on the substrate for which it has the highest Kcat/Km value.
  • Diffusion Limit: For "catalytically perfect" or diffusion-limited enzymes, the Kcat/Km value approaches the theoretical upper limit of 10⁸ to 10⁹ M⁻¹s⁻¹. This means that every time an enzyme molecule collides productively with a substrate molecule, catalysis occurs. The reaction rate is then limited only by the rate of diffusion of substrate to the enzyme's active site.
  • Physiological Relevance: Since cellular substrate concentrations are often below Km, Kcat/Km is a more relevant indicator of enzyme performance in vivo than Kcat or Vmax alone. It represents the rate of product formation when the enzyme is largely unbound and actively seeking substrate.