Let's say that we are screening different enzyme variants, looking for a mutant with increased activity. How would the Michaelis-Menten parameters of the obtained mutant be influenced if low concentrations of the substrate were used during screening?
Thanks!
Hi there,
The Km and kcat are constants specific for an enzyme/substrate system therefore their values don't vary! If working at low substrate concentration ([S]0
If you use very low concentration of substrate in your screening, you are effectively selecting for variants with a high activity at low substrate concentrations. As Dominique pointed out, this can result from either an improvement in Km or kcat, and you can't tell which it is without further characterisation of these parameters. However, if your screening is performed at significantly lower substrate concentrations than your intended use of the enzyme, you risk throwing away potentially interesting mutants. For instance, if a mutant gives you an improved kcat at the cost of a worse Km, it may appear to not be an improvement during the screening conditions, but would in fact be an improvement in your actual process conditions at higher substrate conditions where the Km is less critical.
Dear Daniel,
Please be aware that Michaelis-Menten kinetics does not necessarily reflect any real catalytic activity of an enzyme at LOW CONCENTRATION OF SUBSTRATE. Why? Typically, this kinetics describes cases when the substrate concentration is much greater than KM, the rate of catalysis is equal to kcat, the turnover number. However, most enzymes are not normally SATURATED with substrate. Under physiological conditions, the [S]/KM ratio is typically between 0.01 and 1.0. When [S]
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