For practical purposes, Km is the concentration of substrate which permits the enzyme to achieve half Vmax. An enzyme with a high Km has a low affinity for its substrate, and requires a greater concentration of substrate to achieve Vmax.
The Km of an enzyme, relative to the concentration of its substrate under normal conditions permits prediction of whether or not the rate of formation of product will be affected by the availability of substrate. An enzyme with a low Km relative to the physiological concentration of substrate, as shown above, is normally saturated with substrate, and will act at a more or less constant rate, regardless of variations in the concentration of substrate within the physiological range.
An enzyme with a high Km relative to the physiological concentration of substrate, as shown above, is not normally saturated with substrate, and its activity will vary as the concentration of substrate varies, so that the rate of formation of product will depend on the availability of substrate. If two enzymes, in different pathways, compete for the same substrate, then knowing the values of Km and Vmax for both enzymes permits prediction of the metabolic fate of the substrate and the relative amount that will flow through each pathway under various conditions.
In order to determine the amount of an enzyme present in a sample of tissue , it is obviously essential to ensure that the limiting factor is the activity of the enzyme itself, and not the amount of substrate available. This means that the concentration of substrate must be high enough to ensure that the enzyme is acting at Vmax. In practice, it is usual to use a concentration of substrate about 10 - fold higher than the Km in order to determine the activity of an enzyme in a sample.
If an enzyme is to be used to determine the concentration of substrate in a sample e. Km and Vmax are determined by incubating the enzyme with varying concentrations of substrate; the results can be plotted as a graph of rate of reaction v against concentration of substrate [S], and will normally yield a hyperbolic curve, as shown in the graphs above.
This will enable you to plot a graph of Velocity of reaction absorbance units per sec against Substrate concentration M. From the graph find the maximum velocity and half it i.
Draw a horizontal line from this point till you find the point on the graph that corresponds to it and read off the substrate concentration at that point. This will give the value of Km. Sign up now. Now, you use typical database software to plot the equation. So, knowing the initial rate, Vo, and the various concentration of the substrate, you can create a straight line.
Next, use the reciprocal of the y-intercept to calculate the Vmax of the enzyme activity. Inhibitors alter the maximum rate of the enzyme activity mainly in two ways: competitively and noncompetitively. A competitive inhibitor binds to the activation site of an enzyme blocking the substrate. In this way, the inhibitor competes with the substrate to bind to the enzyme site.
Allowing high concentration of the competitive inhibitor ensures the binding to the site. Hence, the competitive inhibitor changes the dynamics of the enzymatic rate. First, the inhibitor modifies the slope and the x-intercept Km creating a much steeper slope. However, the maximum rate, Vmax, stays the same. On the other hand, a noncompetitive inhibitor binds at a different site than the activation site of the enzyme and does not compete with the substrate.
The inhibitor modifies the structural components of the activation site preventing the substrate or another molecule from binding to the site. This change impacts the affinity of the substrate to the enzyme.
0コメント