Key concepts: Choosing a model

The simplest experiment in enzyme kinetics is to vary the substrate concentration and measure enzyme velocity.

The standard way to fit these data is to fit the Michaelis-Menten model to determine the Vmax (maximum enzyme velocity) and its Km (the concentration of substrate needed to get half-maximal velocity.

The Vmax equals the product of the concentration of active enzyme sites times the turnover rate, kcat. This is the number of substrate molecules each enzyme site can convert to product per unit time. If you know the concentration of enzyme, you can fit the curve to determine kcat and Km. The curve will be identical to the Michaelis-Menten fit.

Many drugs work by inhibiting enzyme activity, either by preventing the substrate from binding to the enzyme, or by stabilizing the enzyme-substrate complex so as to slow formation of product.To distinguish between the models of enzyme inhibition and determine the Ki of the inhibitor, measure substrate-velocity curves in the presence of several concentrations of inhibitor (including one curve with no inhibitor).

Prism can fit your data to three models of enzyme inhibition, plus a more general model which includes the first three as special cases:

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A competitive inhibitor reversibly binds to the same site as the substrate, so its inhibition can be entirely overcome by using a very high concentration of substrate. The maximum velocity of the enzyme doesn't change (if you give it enough substrate), but it takes more substrate to get to half maximal activity. The substrate-velocity curve is shifted to the left but not down.

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A noncompetitive inhibitor reversibly binds to the enzyme-substrate complex, but not to the enzyme itself. This means that the inhibition is not surmountable by increasing substrate concentration, but there is no change in the concentration of substrate needed to get half maximal activity. The substrate-velocity curve is shifted down but neither to the right or left.

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An uncompetitive inhibitor binds with equal affinity to the enzyme, and the enzyme-substrate complex. The inhibition is not surmountable by increasing substrate concentration. Because the enzyme-substrate complex is stabilized, it takes less substrate to get to half-maximal activity. The substrate-velocity curve is shifted down and to the left.

•	The mixed model is a general model that includes competitive, noncompetitive and uncompetitive models as special cases. The model has one more parameter than the others, and this parameter tells you about the mechanism of inhibition.

In some cases, the substrate of an enzyme also inhibits the enzyme by binding to a second site on the enzyme. Prism offers a model to fit substrate-velocity curves when the substrate also inhibits the enzyme.