How I can determine kcat?

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notblue
notblue's picture
How I can determine kcat?

I know that kcat = Vmax/ [Et], but I can’t calculate it. My information available is
Vmax = 0,222 uM/min
Enzyme= 3,2 mg protein/mL and dilutions is: 3,2mg protein /mL x (10/1000) x (20/1000) = 6,4 x 10-4 mg/mL, the MW of my enzyme is 118 000 and it has two active sites.
Thanks for your help

Chin Fen Teo
Chin Fen Teo's picture
Hi notblue,

Hi notblue,

One piece of the information that you are missing is the amount of protein that you actually used in the reaction. It is not clear from your post that whether the dilution that you mentioned is indeed the total amount of enzyme you got in your reaction...

As the unit of kcat is amount of product formation/ (amount of enzyme* time), you can simply divide the Vmax by the amount of protein per reaction.  Says, if your final reaction volume is 1 ml and thus your actually had 6.4x10-4 mg of protein in your tube, then your kcat = 0.222/(6.4x10-4) uM/mg.min.

Furthermore, if you know the molecular weight of your enzymatic product, you can convert the uM into umole, so you can express kcat in term of umole/mg.min, which is a more common use format.

Hope this helps and good luck.

notblue
notblue's picture
 Thanks Pippuri

 Thanks Pippuri

I have a doubt about my enzyme, it has two active sites and I should consider it , how

Chin Fen Teo
Chin Fen Teo's picture
 Hi notblue,

 Hi notblue,

Again, the unit of kcat is defined as "amount of product formation/ (amount of protein*time)", therefore the number of active sites in your enzyme is irrelevant for calculating kcat.

It is only when you need to know the catalytic efficiency of each molecule of your enzyme that the number of active site per enzyme is critical for consideration.

Good luck.