Hi,
I am doing enzyme kinetics of Gtpase and ATPase reaction. In calculating the the parameters I found by plotting the substrate against velocity the enzyme has more Vo values for ATP than GTP. But after plotting the lineweaver Burk plot, the Vmax is greater for GTP than for ATP.
The enzyme is already known to contain ATPase activity but there is null evidence about its Gtpase activity.
Kindly give suggestions.
Thank you.
I would put more trust in the Michaelis (V versus [S]) plot than the Lineweaver-Burk plot. The L-B plot, by using the reciprocals of the measurements, puts the strongest weight for the linear regression on the smallest measurements, which can lead to a large error in the measured parameters. Nonlinear regression analysis of the Michaelis plot is a better method than linear regression with a reciprocal plot.
When calculating the Vmax and Km parameters by nonlinear or linear regression, pay attention to the standard error of the fit parameter values. If the standard errors are comparable in size to the value of the parameters, then the fit should be considered unreliable. This happens if there is a lot of scatter in the data, or if the model (e.g. the Michaelis-Menten equation) is unsuitable.
Thank you for answer.
But I think from michelis Menton plot we can have only theoretical (visual) values of the kinetic parameters without any standard error.
But if not so... I would be thankful if you can guide me over this.
Try using a nonlinear regression computer program to fit your data to the M-M equation (GraphPad Prism, Grafit, and so on). The program will read out the best-fit values of Km and Vmax and the standard error of the fit.
Shivangi .. I concur with Dr Shapiro.
LB plot is notorious for large errors for the smallest of measurements.
Try GraphPad trial. For nonlinear regression, GraphPad has an inbuilt MM model.
To get meaningful conclusion, do get as many data points as possible, preferably >10 since you wish to infer differences between one substrate to another and with data points, the more, the merrier, although this will be a luxury for many enzyme assays.
also refer to
https://www.researchgate.net/post/How-do-I-compare-EC50s-from-fitted-logistic-functions-with-a-t-test
especially to Motulsky's (founder of GraphPad) ideas on how to extract significant difference in nonlinear regression parameters and caveats surrounding this important but seldom discussed issue.
If you are willing to go more advance than consider Monte Carlo or Bootstrap analysis of the Confidence Interval of two MM parameters to signify significance difference or not
Article Evaluation of distribution-Free confidence limits for enzyme...
And GraphPad has Monte Carlo simulation capability.
Yunus Shukor
Thank you for answer.
I am trying grapgh pad for calculation.
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