In zero order kinetics, allowing the assay to run for double time results in double the amount of product. The amount of enzyme present in a reaction is measured by the activity it catalyzes.
The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor.
It is satisfied only when the reaction is zero order. Enzyme activity is generally greatest when substrate concentration is unlimiting.
When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 6. Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first order between B and C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve where the reaction is zero order.
A reaction is most likely to be zero order initially since substrate concentration is then highest. To be certain that a reaction is zero order, multiple measurements of product or substrate concentration must be made. Figure 7 illustrates three types of reactions which might be encountered in enzyme assays and shows the problems which might be enountered if only single measurements are made. Enzymes will work best if there is plenty of substrate.
As the concentration of the substrate increases, so does the rate of enzyme activity. However, the rate of enzyme activity does not increase forever. This is because a point will be reached when the enzymes become saturated and no more substrates can fit at any one time even though there is plenty of substrate available. As the substrate concentration increases so does the rate of enzyme activity.
A continued increase in substrate concentration results in the same activity as there are not enough enzyme molecules available to break down the excess substrate molecules. Introduction to Enzymes Video. Place Order. Introduction to Enzymes The following has been excerpted from a very popular Worthington publication which was originally published in as the Manual of Clinical Enzyme Measurements. Substrate Concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum.
Michaelis developed the following Michaelis constants have been determined for many of the commonly used enzymes. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.
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