Vmax is the maximum rate of an enzyme catalysed reaction i. Km is measure of how easily the enzyme can be saturated by the substrate. Km and Vmax are constant for a given temperature and pH and are used to characterise enzymes. They can be used to identify types of inhibitors i. Vary the catechol concentration to find out Km.
To create a Lineweaver-Burk plot with Prism, use the Transform analysis, then choose the panel of biochemistry and pharmacology transforms. To create a Lineweaver-Burke line corresponding to the nonlinear regression fit, follow these steps:. Create a new XY data table, with no subcolumns. Note the name of this data table. Perhaps rename it to something appropriate.
Go to the Lineweaver-Burke graph. Drag the new table from the navigator and drop onto the graph. Double-click on one of the new symbols for that data set to bring up the Format Graph dialog.
Choose to plot no symbols, but to connect with a line. The product of Kcat times Et the concentration of enzyme sites equals the Vmax, so if you know Et, Prism can fit kcat.
That allosteric model adds an additional parameter: the Hill slope h. When h equals 1. The rate of formation of product now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of the reaction to any significant effect. The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax. The relationship between rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate.
This is usually expressed as the Km Michaelis constant of the enzyme, an inverse measure of affinity. 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.
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