Hi researchers…
Is it true that calculate kcat of my enzyme with this formula? Kcat=Vmax/km
I saw this formula in an article: Kcat=Vmax/[E]; but I don’t understand which enzyme concentration?! Total enzyme concentration???
In brief, yes: Vmax=[E]tot*kcat. You can find detailed explanations in books (from Lehninger's Biochem to Cornish-Bowden's Enzyme kinetics) or https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics
Yes Kcat=Vmax/[E], where [E] = total enzyme, i.e., free enzyme and enzyme bound to substrate or intermediate.
Hi Farhadi,
It's true that to calculate Kcat of an enzyme , you can use Kcat=Vmax/[Et].However, to calculate [Et]=Total enzyme conc, you need the amount of your protein and the total volume of the enzymatic reaction.For example, if X ul of your reaction contains Yug of your enzyme, how many ug of your enzyme will be present in 1L(10^6ul)........=(10^6ul x Xug)/Yul. Therefore, to determine the total conc[Et] in uM will be the amount of your enzyme(ug)/MW of your enzyme in Dalton. Note that if your enzyme MW is in kDa(x1000).
I hope this will help you.
All the best!
Hi Farhadi
The replies are all perfectly good but they have not pointed out that as the actual concentration of the enzyme protein must be known, you need to be sure that you are able to calculate the concentration using the molecular weight and the purity of the enzyme. It the sample is impure or part of a tissue extract for example, much of the protein present in the assay mixture will not originate from your enzyme.
Peter
Peter J Butterworth makes an important point, one should add that manufacturers often only claim minimal purity, the actual purity is usually larger, and, to make matters worse, will change over time. So even a batch analysis, if you can get it, doesn't really help.
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