From the graph find the maximum velocity and half it i. Draw a horizontal line from this point till you find the point on the graph that corresponds to it and read off the substrate concentration at that point. This will give the value of Km. Sign up now. Determining Km and Vmax. On a plot of Velocity versus Substrate Concentration V vs. It should be noted that the value of V max depends on the amount of enzyme used in a reaction. Double the amount of enzyme, double the Vmax. 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 Vmax 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.
Km and Vmax are determined by incubating the enzyme with varying concentrations of substrate; the results can be plotted as a graph of rate of reaction v against concentration of substrate [S], and will normally yield a hyperbolic curve, as shown in the graphs above. The relationship is defined by the Michaelis-Menten equation:. I t 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:. This is the most widely used method of linearising the data, and generally gives the best precision for estimates of Km and Vmax.
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. The Eadie-Hofstee plot rearranges the Michaelis-Menten equation as:. 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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