Unlocking the Key to Enzymes: Studying Enzyme Kinetics

By virtue of its quantitative nature, NMR spectroscopy is increasingly becoming the method of choice to monitor a reaction and determine its kinetic parameters. We’ve demonstrated the ability of the NMReady-60 to monitor a reaction and subsequently extract kinetic parameters in a previous blog post. In this blog post, I’d like to show how the NMReady-60 can be used to study enzyme kinetics. Adapted from a Journal of Chemical Education article published by Olsen and Giles,[1] the enzymatic hydrolysis of N-acetyl-DL-methionine by porcine acylase was studied.

Figure 1.  Stacked plot of 1H NMR spectra of the hydrolysis of  N -acetyl-DL-methionine by porcine acylase to produce L-methionine.

Figure 1. Stacked plot of 1H NMR spectra of the hydrolysis of N-acetyl-DL-methionine by porcine acylase to produce L-methionine.

As seen in figure 1, the 1H NMR spectrum of the hydrolysis reaction shows the depletion N-acetyl-DL-methionine (4.25 ppm, red dot) and the simultaneous appearance of the product, L-methionine (3.85 ppm, blue dot). Since the porcine acylase selectively hydrolyzes N-acetyl-L-methionine, the signal at 4.25 ppm never completely disappears because the D-enantiomer of the racemic mixture remains in the solution. From the integration data, the plot of N-acetyl-L-methionine concentration over time (figure 2) can be generated.

Figure 2.  Plot of  N -acetyl-L-methionine concentration over time of the reaction.

Figure 2. Plot of N-acetyl-L-methionine concentration over time of the reaction.

With the concentration data in hand, the reaction rate at different points can be approximated from which both the Michaelis-Menten (figure 3) and Lineweaver-Burk (figure 4) plots could be constructed.

Figure 3.  Michaelis-Menten plot of the reaction.

Figure 3. Michaelis-Menten plot of the reaction.

From the Lineweaver-Burk plot, the values of Vmax (0.3152 mmol L-1 s-1) and KM (0.24 mol L-1) were extracted. These values are related to enzyme activity and crucial to understanding the behaviour of the enzyme being studied. The experiment can be easily adapted to study other enzymatic reactions and serves an excellent introduction to enzyme kinetics and how NMR spectroscopy can be used to study them. For more information on the experiment, please check out our recently published application note.

Figure 4.  Lineweaver-Burk plot of the reaction.

Figure 4. Lineweaver-Burk plot of the reaction.

References
[1]Olsen, R., Olsen, J. and Giles, G. (2010). An Enzyme Kinetics Experiment for the Undergraduate Organic Chemistry Laboratory. Journal of Chemical Education, 87(9), pp.956-957.