Enzyme kinetics by real-time quantitative NMR (qNMR) spectroscopy with progress curve analysis.

This review article summarizes how the experimental data obtained using quantitative nuclear magnetic resonance (qNMR) spectroscopy can be combined with progress curve analysis to determine enzyme kinetic parameters. The qNMR approach enables following the enzymatic conversion of the substrate to the product in real-time by a continuous collection of spectra. The Lambert-W function, a closed-form solution to the time-dependent substrate/product kinetics of the rate equation, can estimate the Michaelis-Menten constant (KM.) and the maximum velocity (Vmax) from a single experiment. This article highlights how the qNMR data is well suited for analysis using the Lambert-W function with three different applications. Results from studies on acetylcholinesterase (acetylcholine to acetic acid and choline), ß-Galactosidase (lactose to glucose and galactose), and invertase (sucrose to glucose and fructose) are presented. Furthermore, an additional example of how the progress curve analysis is applied to understand the inhibitory role of the artificial sweetener sucralose on sucrose's enzymatic conversion by invertase is discussed. With the wide availability of NMR spectrometers in academia and industries, including bench-top systems with permanent magnets, and the potential to enhance sensitivity using dynamic nuclear polarization in combination with ultrafast methods, the NMR-based enzyme kinetics could be considered a valuable tool for broader applications in the field of enzyme kinetics.

NMR based real-time enzyme kinetics on estimating the inhibitory effect of sucralose in the enzymatic conversion of sucrose.

Sucralose, one of the popular non-caloric artificial sweeteners, has been known to influence the enzymatic conversion of sucrose to glucose and fructose by invertase. In continuing the use of real-time NMR experiments and reaction progress curve analysis to measure enzyme kinetics, here we investigate the role of sucralose as an inhibitor. NMR based kinetic experiments were performed as a function of the substrate concentration for a range of sucralose concentrations, and the results were analyzed by fitting the progress curve to the Lambert-W function. The Michaelis-Menten parameters were then used to estimate the inhibitory constant of sucralose. To estimate the extent of sucralose inhibition on the enzymatic production of glucose, control experiments were performed with lactose as the inhibitor under similar experimental conditions. The results show that sucralose is a much more potent inhibitor than lactose, inhibiting the enzymatic conversion at least seven times more.