[diablo]

Hi, i got a prob to solve this:

A enzyme requires 2 substrates (a saccharide + GDP-fucose).

In order to obtain a Km value the GDP-fucose concentration was varied
and, after incubation for 30 min in presence of enzyme+1 nmol saccaride (volume 10µl) the saccharide substrate + productwere seperated by HLPC following data ware obtained:

PAS   8126222  10468052  7106807  9566786
PAP     162834    273438     206554   306619
GA       0,2 nmol  0,4nmol     0,6nmol    0,8nmol

PAS... PEak area of remaining substrate
PAP... Peak area of product
GA.... GDP-Fucose amount (initial)

now i should calculate the Km value of the enzyme for GDP-Fucose
Hint: calculate the concentration of GDP-fucose + amount in nmol of product (based on the ratio of product/product+substrate multiplied by 1 nmol) Then calculate Substrate + V


Now my prob: i dont how to appraoch to this problem... --> i guess that i have to plot something, but what ? --> and how can i calculate the concentration (hint)?

Thx for help,
Diabloooooo

[Yggdrasil]

Since you're dealing with enzyme kinetics, you want to use the Michaelis-menten equation.  Specifically, if you rearrange the MM equation, you can obtain the Lineweaver-Burke equation.  This equation tells you that if you plot the reciprocal of initial rate of product formation against the reciprocal of substrate concentration, you can determine the Km of the substrate:

http://en.wikipedia.org/wiki/Lineweaver-Burk_plot

[diablo]

thx man !