Biology – Factors that affect enzyme action | e-Consult

Determining V_max Experimentally:

1. Reaction Conditions: The enzyme-catalyzed reaction is carried out with a range of substrate concentrations (S) held constant, while other factors like temperature, pH, and enzyme concentration are kept constant.
2. Rate Measurement: The initial rate of the reaction (v) is measured for each substrate concentration. This is typically done by monitoring the disappearance of substrate or the appearance of product over time.
3. Plotting the Data: The initial rate (v) is plotted against the substrate concentration (S). This results in a hyperbolic curve.
4. V_max Identification: The maximum rate of reaction (V_max) is the plateau that the curve approaches as the substrate concentration increases. It represents the rate at which the enzyme is working when all active sites are saturated with substrate. The V_max value is determined by extrapolating the hyperbolic curve to the y-axis. This is often done graphically or using non-linear regression analysis.

Relationship between V_max and Km:

The Michaelis-Menten equation describes the relationship between initial reaction rate (v), substrate concentration (S), Km, and V_max:

v = (V_max * S) / (Km + S)

Km as an indicator of affinity:

The Michaelis-Menten constant (Km) is defined as the substrate concentration at which the reaction rate is half of V_max (v = V_max/2). Therefore, Km is inversely proportional to the affinity of the enzyme for its substrate. A low Km indicates high affinity – the enzyme achieves half-maximal velocity at a low substrate concentration. Conversely, a high Km indicates low affinity – the enzyme requires a high substrate concentration to achieve half-maximal velocity. The lower the Km value, the stronger the interaction between the enzyme and its substrate.