Using enzyme extinction curves to plot Km and Vmax - (Oct/05/2006 )

Hi, I am new to molecular biology and want to ask advice on this subject.


I have an enzyme assay where, once I give the enzyme mixture the substrate, a colour change can be observed at Xnm giving a negative slope. I have absolutely no idea how to use this slope to calculate Km, Vmax etc. I have checked the web, but all I can find is plots of product vs time?

ANy help would be much appreciated.
cheers

Graham

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you should do your experiment with different substrate concentrations over a given time or to a certain time-point; for each experiment you have to determine the specific enzyme activity; if you have some substrate points, some below and some beyond roughly estimated Km, you can determine precise Km and Vmax; use a fitting program such as Grafit or newer versions of Sigmaplot

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you should do as kosmodrome says and perform a substrate saturation curve set of assays. you can then use the results to make a "lineweaver-burk" plot (1/v vs 1/s). the x intercept is -1/Km and the y intercept is 1/Vmax. see this wiki page:

lineweaver-burk plot

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I am still a bit fuzzy here. When I do say, 100mM of a substrate i get a slope of say - 0.03. When I do 200mM I get say -0.09. Do I then plot the slopes againt the substrate concs? I am just not sure how to go about this at all??!

cheers

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slope should express the linearity of enzyme activity over time and conditions; classically, slopes should be expressed in specific enzyme activity for final presentation, especially to express Vmax correctly;

to your questions: you can plot slopes against substrate conc.; for Lineweaver burk plot use reciprokes as suggested; you ease your life by using a PC program; there are a lot around to determine Km and Vmax

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your enzyme assay is converting a component (nadh or nadph?) from one that absorbs at your reading wavelength to one that does not absorb or absorbs less. the direction of the slope is then negative. in this case the direction doesn't matter, just the rate of change matters. you can equate the rate of change to the moles of substrate (or cofactor) converted per unit time using the extinction coefficient of the absorbing species. or you can just use the absolute change in absorbance per unit time for v.

so, in answer to your question, you can plot the reciprocals of absolute slope vs substrate concentration for the lineweaver-burk plot.

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