While some of the presentation may seem somewhat dated, the basic concepts are still helpful for researchers who must use enzymes but who have little background in enzymology. 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.
This is represented graphically in Figure 8. It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax.
Using this maximum velocity and equation 7 , Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. This is shown in Figure 8. Using this constant and the fact that Km can also be defined as:. B As the concentration of substrate increases, the enzyme becomes saturated with substrate.
As soon as the catalytic site is empty, more substrate is available to bind and undergo reaction. 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.
The ES complex is formed by combining enzyme E with substrate S at rate constant k 1. The ES complex can either dissociate to form E F free enzyme and S, or form product P at rate constant k 2 and k 3 , respectively.
The velocity equation can be derived in either of the 2 methods that follow. E, S, and the ES complex can equilibrate very rapidly. The total enzyme concentration E T is equal to the concentration of free enzyme E E F plus the concentration of the bound enzyme in ES complex:. The figure above shows the relatively low and constant concentration of the enzyme-substrate complex due to the complex's slow formation and rapid consumption. Note the falling substrate concentration and the rising product concentration.
The rates of formation and breakdown of the E - S complex are given in terms of known quantities:. They typically range from 10 -1 to 10 -7 M. This means that the rate and the substrate concentration are directly proportional to each other.
The reaction is first-order kinetics. This means that the rate is equal to the maximum velocity and is independent of the substrate concentration. The reaction is zero-order kinetics. Figure 2 : Diagram of reaction speed and Michaelis-Menten kinetics.
0コメント