How to use it
- Paste the substrate and *initial rate* columns. The initial rate is the slope of the first straight part of a progress curve.
- Spread the substrate concentrations either side of Km — from about 0.2 × Km to past 5 × Km where the experiment allows it.
- Check the highest substrate in multiples of Km in the result. Below 5 × Km there is a warning, and that Vmax should not be used as it stands.
- Look at whether the points flatten at the right-hand edge of the plot. If they are still climbing, the substrate needs to go higher.
💡 Any units work — Km comes back in your concentration units and Vmax in your rate units.
Formula and cautions
Rate rises with substrate until the enzyme is saturated and it rises no further. Writing that shape down as two numbers is the Michaelis–Menten equation.
The two parameters
Vmax is the rate at infinite substrate; Km is the substrate concentration at which the rate is half of it.
- Rate
- v = Vmax · [S] / (Km + [S])
- Km, by definition
- v(Km) = Vmax / 2
Vmax / Km
The slope of the curve at vanishing substrate. It is the better single number for comparing enzymes or substrates, because it summarises a change in either parameter where Km or Vmax alone would each tell half the story.
Why Km is always positive
Noisy points at low substrate can make a negative Km arithmetically cheaper. A negative Km is not a rate constant, though — it is the search leaving the model — so both parameters are held positive.
Cautions
- ⛔ If they are not *initial* rates, the equation does not apply. End-point readings, or readings taken after the reaction has slowed, still produce a curve and a Km and neither means anything. No arithmetic can detect this.
- Without substrate well above Km, Vmax is set by the model rather than by the data. That is why the highest substrate is always reported in multiples of Km.
- Km is not a fixed property of an enzyme; it is the value under those conditions. pH, temperature, ionic strength and buffer all move it. Compare conditions before comparing numbers.
- A high R² does not mean the model is right. Whether the points scatter evenly along the curve matters more.
- The standard errors say how tightly your points pin Km and Vmax down. How reproducible the assay is, is a separate question that only replicates answer.
Frequently asked questions
Often used together
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