Hi Umair,

You could use the GraphPad Prism (v8). This is the best software to analyze kinetics including LB plots. You can download free and access from the link: http://www.download82.com/download/windows/graphpad-prism/

Best of luck.

Manju

To perform the Lineweaver-Burk plot, you need to make new cells containing the reciprocal of the rate and the substrate concentration. Then make a scatter graph plotting 1/rate on the y-axis and 1/[substrate] on the x-axis.

However, the Lineweaver-Burk plot is not a good method for determining the inhibitory mechanism. It looks great with perfect data. I will make a confident prediction that your data are not perfect, because I don't know anyone who gets error free data. Lineweaver-Burk plots are especially bad as the least accurate point (lowest [substrate]) has the highest weight in a double reciprocal. Athel Cornish-Bowden and others have published ad nauseum on the issues of the Lineweaver-Burk plot (see e.g. https://www.wiley.com/en-gb/Fundamentals+of+Enzyme+Kinetics%2C+4th+Edition-p-9783527330744 , an excellent book on enzyme kinetics). Basically, relatively modest errors in an experiment like yours makes the Lineweaver-Burk plot equivocal between at least two of the mechanisms.

A much better approach is to fit your data to the various equations using statistical software, and find which equation fits best to the data. If you have access to GraphPad, this will work (you'll need to use the online help pages as this is right at the limit of GraphPad's capability). Alternatively, the R statistical software is free and extremely powerful, if you don't mind coding. I have written scripts for R for exactly this problem and could share directly if you have R set up. The approach is explained in detail in my chapter on enzyme kinetics ( Chapter Reaction Chemical Kinetics in Biology

Chapter Reaction Chemical Kinetics in Biology

Thank you so much guys.

I agree with Nicholas J Harmer. You will get your answer as per his suggestion

Please accept Manju's answer. Data should be fitted to the Michaelis-Menten equation using non-linear regression methods. GraphPad has all you need for this. The fitting will provide values for Km and Vmax in the presence/absence of inhibitor and so reveal the type of inhibition. Once you have computed values for the kinetic constants you can display the results with a Lineweaver-Burk plot with data points fitted by a line derived from the computed values for Km and Vmax. Data collected for Dixon plots and/or Cornish-Bowden plots should also be examined. Non-linearity of such plots will be indicative of complex behaviour e.g. binding of more than one molecule of inhibitor to a site on the enzyme.

Peter

Thank you Peter J Butterworth. I highly appreciate your response. I am almost done with that part but I am facing problem with plotting lineweaver burk plot. Any video tutorial link.

The LB-plot is useful for the presentation of data, as most biologist can recognise the patterns obtained by different forms of inhibition. However, it should not be used to calculate the inhibition constants, because any linearisation severely biases the results.

Instead, use curve fitting to the entire data set by Marquardt-Levenberg (http://137.204.42.130/~bittelli/materiale\_lettura\_fisica\_terreno/marquardt\_63.pdf) or - even better - simplex (doi:10.1093/comjnl/7.4.308).

Alternatively, use the direct plot (http://www.biochemj.org/bj/139/0715/1390715.pdf), this method is particularly useful if the data contain a lot of scatter, due to the use of medians.

I have discussed enzyme inhibition in detail in doi:10.1007/978-3-319-19920-7_6, this chapter also contains a fully worked-out example for the use of direct plots.

Some tutorial links could be of some help.

https://www.youtube.com/watch?v=5VL9Y8cXFCQ

https://www.youtube.com/watch?v=UTeE5f9Ai-A

I agree with the explanation by Peter J Butterworth. Also Engelbert Buxbaum has given great pointers, constants should only be estimated by non-linear curve fitting using LM or such iterative fitting methods. Similar workflows have been used in our recent paper, it might be of some help to your work.

https://www.sciencedirect.com/science/article/pii/S0308814619304777#s0010

All the best.