Enzyme parameters

In this exercise you will perform an experiment in which you measure the enzyme-catalysed reduction of an oxidized substrate into a reduced product. Your aim is to determine Km of an enzyme from the reaction of the enzyme at different substrate concentrations. The scheme of the reaction is shown below.

Note - You measure the rates of the reaction indirectly by following the consumption of NADH, not the consumption of the substrate itself.

1. Measure the rate of the blank reaction, ie without enzyme. To do so, click the "Start"-button. This will stmeasure a new substrate concentration.

2. Select a substrate concentration (e.g. 5 µM) by clicking on the respective button below. Start the reaction by clicking on the "Start"-button again. At convenient time intervals (for example every 5 seconds) write down the corresponding absorbance. Do so until you have recorded at least 6 measurements. When you have finished, stop timer and reaction by clicking on the 'Stop'-button. Make sure you then also click on the "Reset"-button when you start a new reaction.

3. Repeat step 2 for all substrate concentrations. Make sure you keep track of the tested substrate concentrations.

4. Determine the enzyme parameters KM and Vmax using appropriate methods.