NMR for direct determination of K-m and V-max of enzyme reactions based on the Lambert W function-analysis of progress curves

作者：Exnowitz Franziska; Meyer Bernd; Hackl Thomas^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2012, 1824(3): 443-449.

DOI：10.1016/j.bbapap.2011.10.011

• H-1 NMR
• Enzyme kinetics
• Progress curve analysis
• Lambert W function
• lnvertase
• Germacrene D synthase

H-1 NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme%26apos;s kinetics, K-m and V-max, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the lambert W function the parameters K-m and V-max were fitted to obtain the experimental progress curve and resulted in K-m = 28 mM and V-max = 13 mu M/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K-m = 379 mu M and k(cat) = 0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.

• 出版日期2012-3