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Measuring KM and Vmax

Measuring KM and Vmax

The quantities KM and Vmax are experimentally determined and different for each enzyme. Once you have an assay for enzyme activity, you can determine these parameters.

You can estimate KM and Vmax from the graph of initial velocity versus [S].

  1. Run a series of reactions with constant [Etot], varying [S], and measure Vo.
  2. Graph Vo vs. [S].
  3. Estimate Vmax from asymptote.
  4. Calculate Vmax/2
  5. read KM from graph.
Vmax is difficult to determine if data is graphed this way, since the graph is hyperbolic. It is hard to extrapolate to infinite [S] and guess Vmax.

For this reason, the Lineweaver-Burke plot is used.
You can rearrange (6) into linear form: y=mx+b, if you let and .

Then (6) rearranges to:

If the data is plotted in this way, it looks like:

From this type of graph, it is easy to estimate KM and Vmax.

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