How to Find Km and Vmax

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shafqat
shafqat's picture
How to Find Km and Vmax

Hi,
I am working on enzyme kinetics and want to discuss some issues like:-
1) What shuld be the experimental strategies?
2) what are the purification states we can use for the study of enzyme in question? Can we use the cellular extract directly or we have to purify the enzyme then only assay will be possible?
 Please lets discuss.

Jason King
Jason King's picture
If you have a source of

If you have a source of substrate you can add and you can measure the product of the reaction then you should be able to calculate Km and Vmax without having to purify your enzyme. Enzyme kinetics are usually done using cell extracts.

shafqat
shafqat's picture
parvoman wrote:

parvoman wrote:

If you have a source of substrate you can add and you can measure the product of the reaction then you should be able to calculate Km and Vmax without having to purify your enzyme. Enzyme kinetics are usually done using cell extracts.

Thanx for your reply. I am working on an extracellular enzyme "chitinase". I am having source of substrate. So, I will go like this:-
1) I will pellet down cells and collect the supernatant.
2) I will set the reaction with substrate and supernatant.
3) compare the ODs with standard curve.
4) Plot the graph for Km and Vmx
If any further suggestions plz reply.

Shubhangi
Shubhangi's picture
Enzyme substrate reaction is

Enzyme substrate reaction is very specific hence your strategy should work fine. Just make sure there are no inhibitory substances in supernatent.
different concentrations of substrate and dilutions of sup can be included for getting more data.
use proper controls, blanks