Double the amount of enzyme, double the V max. If one wanted to compare the velocities of two different enzymes, it would be necessary to use the same amounts of enzyme in the different reactions they catalyze. It is desirable to have a measure of velocity that is independent of enzyme concentration. For this, we define the value Kcat , also known as the turnover number. To determine Kcat , one must obviously know the V max at a particular concentration of enzyme, but the beauty of the term is that it is a measure of velocity independent of enzyme concentration , thanks to the term in the denominator.
K cat is thus a constant for an enzyme under given conditions. This would mean that each molecule of enzyme is catalyzing the formation of 35 molecules of product every second. This astonishing number illustrates clearly why enzymes seem almost magical in their action. Another parameter of an enzyme that is useful is known as Km , the Michaelis constant.
What it measures, in simple terms, is the affinity an enzyme has for its substrate. Affinities of enzymes for substrates vary considerably, so knowing Km helps us to understand how well an enzyme is suited to the substrate being used. Measurement of Km depends on the measurement of V max. The catalytic site of the enzyme is empty, waiting for substrate to bind, for much of the time, and the rate at which product can be formed is limited by the concentration of substrate which is available.
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. 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.
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