Enzyme Kinetics

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doriskarloff60
doriskarloff60's picture
Enzyme Kinetics

I have some work due in at my univeristy and am having real difficulty understanding how to calculate Vmax, Km and Kcat. I've been given a graph, see attahced, but the data is not clear enough to plot an accurate lineweaver-burk graph. Is there any other way to calculate these values?
Any help much appreciated.
 

Jason King
Jason King's picture
doriskarloff60 wrote:

doriskarloff60 wrote:

I have some work due in at my univeristy and am having real difficulty understanding how to calculate Vmax, Km and Kcat. I've been given a graph, see attahced, but the data is not clear enough to plot an accurate lineweaver-burk graph. Is there any other way to calculate these values?
Any help much appreciated.
 

 
You should be able to read off the Vmax by placing a ruler horizontally between the top of the curve and the y-axis. This looks possible for one of the curves but with the other it doesn't look like enough substrate concentrations have been tested to accuately say where the rate levels off. It says in the details below the plot how long the reaction was followed - you need this and the y-axis value to calculate the reaction rate.
Km should be the substrate concentration at half of the Vmax - again, read it off where possible.
 
The rests depends on being able to calculate these two values.

PhilM
PhilM's picture
They really did not want to

They really did not want to help you out with accurate information there did they?
I scaled your PDF graph and so I could easily use a ruler to esimate the values
[Peptide A] (mM)           hydrozyled (pmol)
0.1                                     1
0.3                                     4
0.6                                   6.5
1.2                                     9
2                                      8.2
4                                      9.2
6                                      9.8
[Peptide B] (mM)           hydrozyled (pmol)

0.01                                  1.5
0.02                                   2
0.03                                  3.2
0.05                                    5
0.075                                 5.2
1.5                                     6.2
3                                        7.4
I don't know if you are allowed to use graphing software for your problem set but I did a one site bind non-linear regression (y = Vmax * x / (Km + x)) and got the following values
Enzyme with peptide A
Parameter    Value    Error
----------------------------------------
Vmax    10.60649    0.68622
Km         0.43845      0.11381
----------------------------------------
Enzyme with peptide B

Parameter    Value    Error
----------------------------------------
Vmax    7.05708    0.43518
Km           0.0328    0.00722
 
For the kcat value - they tell you the molecular weight of enzyme is 78000, and you have 0.8µg in 20µl - so [E]0 = 51.3µM
If [S] is large compared to KM then the term [S]/(Km+[S]) ≈ 1, and the rate of product formation is d[P]/dt ≈ Vmax = Kcat*[E]0

so you can estimate Kcat as Vmax/[E]0