Hot calculate Kcat?

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mtbazz
mtbazz's picture
Hot calculate Kcat?

Can someone please explain to me how to calculate kcat from a Km curve ?

The information I have available to me is the Km and Vmax, the molecualr weight of my enzyme, and the slope from the std curve of my product.

carlahee
carlahee's picture
kcat is just the Vmax divided

kcat is just the Vmax divided by the enzyme concentration you used to do the experiment. The units end up being 1/sec.

Hope this helps!

mmk
mmk's picture
[snip]

[snip]
The information I have available to me is the Km and Vmax, the molecualr weight of my enzyme, and the slope from the std curve of my product.

What are the units of your Vmax values?

If your Vmax is simply a raw rate (e.g. uMolar/min), then you need first to convert it to Specific Activity (SA) by dividing by the amount of enzyme in your assay to give uMolar/min/mg. Remember not to get confused here between Molar and moles. In this example the amount of enzyme is in ug so it's just a simple division. Then divide your SA by the molecular weight of your enzyme to give kcat (1/min). Divide again by 60 to give kcat (1/sec).

Tirumal Rao
Tirumal Rao's picture
Competitive: Km(with) = Km

Competitive: Km(with) = Km(without)*(1+([I]/Ki))

Non-competitive: Vmax(with) = Vmax(without)/(1+([I]/Ki))
The ambitious student may also calculate the inhibitor constant, Ki, for the inhibitor, which is a measure of how strongly the inhibitor is bound. The value of Ki corresponds to the inhibitor concentration when half of the enzyme molecules bind an inhibitor molecule (half-saturation concentration). If the inhibition is pure competitive or non-competitive, then Ki is the same as the dissociation constant, Kd, for the enzyme-inhibitor complex. The relationship between the Vmax and Km values in the absence and presence of the inhibitor are different for the two types of inhibition
You may also look up some published data on the beta-galactosidase enzyme. In the abstract for the reference Tanaka et al (1975) you will find the Km value for a related substrate, oNP-Gal. Search for the article in Medline http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
Regards,
Tirumal
India
Tirumal

luweidong
luweidong's picture
 1, You sould change V (μmol

 1, You sould change V (μmol/min) to μmol/min/mg. eg: (Vmax=100U/mg=100μmol substrate/(1mg enzyme min)=100μmolsubstrate/{(0.001g/MWDa)*1000000 min-}=????
2,[E]t=1000/MW (kda)=????
3,Kcat=Vmax/[ET]= answer that you want.

Janet Gonzalez
Janet Gonzalez's picture
which is the best and easiest

which is the best and easiest software to do calculate Km Vmax and Ki?

mustafa12
mustafa12's picture
I really need some help with

I really need some help with this question!
The initial rate of an enzyme reaction is calculated in Absorbance/minute.
This is then converted to micromoles substrate oxidised per minute using the molar extinction coefficient of NADH which is 6.22 x 103 (i.e a molar solution of NADH in a 1cm light path has an absorbance of 6.22 x 103)
Now....Calculate the activity of the enzyme in micromole/min/mg???

Ive already converted the initial rate of the enzyme reaction to micromoles of substrate oxidised per min using the beer lambert law.....but i now cannot calculate the activity of the enzyme.
Could anyone PLEASE give a detailed explanantion of how to do it....

mohit sona
mohit sona's picture
kcat calculation

I found this information about kcatV= Kcat [Enzyme] [S] / (Km + [S]) Anna Lezhneva