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I have some adoubt on Km calculation. I have some data as follows to calculate Km value of Midazolam:
Conc. (uM): 0.6, 1, 1.5, 3, 6, 9, 15 and 30; the corresponding V is: 0.94, 1.90, 2.63, 4.21, 6.52, 7.64, 8.23 and 8.45.
If I use Lineweaver-Burk plot to calculate, the Km value is about 9.4 uM (the r2 is about 0.97, not very good), but if I use Nonlin fit method of Prism to calculate, the Km value is about 3.6 uM. From the view of Lineweaver-Burk plot, the data of 0.6 uM is not good, so I exclude this data to re-calculate. The r2 can reach 0.998 and the Km value is about 4.8 uM; but the Km value using Nonlin fit method to calculate is about 3.7 uM;
And if I also exclude the data of 30uM, the r2 can be optimized furtherly to reach 0.9992, and the Km is about 5.1 uM, no very much difference compared with above, but the Km value got from Prism under this condition is elevated to 4.6 uM. So I wonder which data is the exact Km value of midazolam? 3.7 or 4.8 or 5.1 uM? What is the strategy we should obey when we calculate Km values? Any suggestions are welcome. Thanks!
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Nowadays, most enzymologists agree that the Lineweaver-Burk plot should definitely NOT be used for accurate determination of Km.
Non linear fitting methods should be definitely preferred. Before non linear regression is performed (i.e., before selection of the equation for data fitting), the Eadie-Hofstee plot should be examined to check for possible involvement or more than one enzyme or for atypical kinetics (in such cases, the Michaelis-Menten equation might not be the best equation that describes the data).
First, let me hypothesize that you may be worrying too much: given the variability inherent to any enzyme kinetics experiment, I'd say that 3.7 uM and 5.1 uM are not really different.
In the case you describe, one can expect some uncertainty due to limited amount of data (n=1 per level, 8 levels).
Duplicate measurements would have provided better precision (16 data points instead of 8 -in such case, do not run non linear regression on mean values).
In my humble opinion, 3.7uM seems to the best Km value that can be computed from the data set you obtained (non linear regression, no data point excluded). However, a number of things may be checked before drawing such conclusion, such as:
- the V values should be actually initial reaction rate values, or at least as close to as technically possible. - the V values should refer to metabolite formation rather than substrate disappearance - The concentration values adequately encompass the expected value of Km; however the highest value is only about 5-6 times as high as expected value of Km; does it really approach saturation (V close to Vmax)?
- Check the regression itself: not only r2, but also standard error on Km and Vmax; check the distribution of residuals; how does the curve recalculated with found parameters pass through the data points...
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The Km of Midazolam is around 5 uM. Midazolam is a CYP3A4 substrate that follows the CYP3A4 multi-site binding, heterotropic cooperativity kinetics that is exhibited by 3A4 substrates. Therefore your data of 3.5-5 uM are all within 2 fold of Km value of Midazolam and valid.
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