This program fits initial rate data for enzyme catalyzed reactions to the equation below using the method of Wilkinson, G.N. (1961) Biochem. J. 80, 324-332.
v(obs)=Vm.So/(So+Km)
Data can be inputted and fitted as initial rates. Fitted values of Vm, Km and Vm/Km are outputted. If the enzyme concentration is known it can be inputted and values of kcat and kcat/Km calculated.
Data can also be inputted in the form of Abs/min and if the molar extinction is inputted then the Abs/min will be converted to d[P]/dt=v(obs) in M/min. Data can then be converted to M/sec and fitted. Initial rates are often very small numbers and so to ensure numerical accuracy you can multiply your data by the scaling factor of your choice before fitting the data.
Or the initial rates can be divided by the enzyme concentration to give kcat(obs) which can then be fitted.
When fitting without estimates, a linear Lineweaver-Burk plot is used to make the initial estimate of kcat and Km. In this case usually, one iteration using the Wilkinson method usually gives the best fit. Please note the Lineweave-Burk method is only used to make the initial estimate, the final outputted fit is always by the Wilkinson method.
If the initial Lineweaver-Burk plot does not give good estimates of kcat and Km you can enter your own estimates and then fit by the Wilkinson method using your own estimates of kcat and Km. Doing more iterations does not necessarily improve the fit obtained.
Results can printed, saved as a text document or copied and pasted into other applications such as Word.
Instructions and test data are provided.
If your data is in a simple two column excel file and if you save it as a csv file then MMfit should be able to read the file and analyse the data.
You may also be interested in the iPhone app EnzymeQA as it contains key facts in enzymology and should help you check that you have used the appropriate conditions for determining catalytic constants.
