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Mikales Menten Great With Green Book Also
Mikales Menten Great With Green Book Also
A simple chemical reaction with a single substrate shows a linear relationship between the rate of
formation of product and the concentration of substrate, as shown below:
For an enzyme-catalysed reaction, there is usually a hyperbolic relationship between the rate
of reaction and the concentration of substrate, as shown below:
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(A) At low concentration of substrate, there is a steep increase in the rate of reaction with increasing
substrate concentration. 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.
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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."
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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
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varies, so that the rate of formation of product will depend on the availability of substrate.
v = Vmax / (1 + (Km/[S]))
It is difficult to fit the best hyperbola through the experimental points, and difficult to determine Vmax
with any precision by estimating the limit of the hyperbola at infinite substrate concentration. A number
of ways of re-arranging the Michaelis-Menten equation have been devised to obtain linear
relationships which permit more precise fitting to the experimental points, and estimation of the values
of Km and Vmax. There are advantages and disadvantages associated with all three main methods of
linearising the data.
The Lineweaver-Burk double reciprocal plot rearranges the Michaelis-Menten equation as:
y intercept = 1 / Vmax
gradient = Km / Vmax
x intercept = -1/ Km
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This is the most widely used method of linearising the data, and generally gives the best
precision for estimates of Km and Vmax. However, it has the disadvantage of placing
undue weight on the points obtained at low concentrations of substrate (the highest values
of 1/[S] and 1/v). These are the points at which the precision of determining the rate of
reaction is lowest, because the smallest amount of product has been formed.
v = Vmax - Km x v / [S]
y intercept = Vmax
gradient = -Km
x intercept = Vmax / Km
This plot overcomes the problem of uneven spacing of points, and undue weight given to points
at low concentrations of substrate. However, it has the disadvantage that v, which is a
dependent variable, is used on both axes, and hence errors in measuring the rate of reaction are
multiplied, resulting in lower precision of the estimates of Km and Vmax
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y intercept = Km / Vmax
gradient = 1 / Vmax
x intercept = -Km
This plot overcomes the problem of uneven spacing of points, and undue weight given to points
at low concentrations of substrate. However, it has the disadvantage that [S] is used on both
axes, and hence pipetting errors, which lead to errors in the true concentration of substrate
available, are multiplied, resulting in lower precision of the estimates of Km and Vmax.
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