Quantitative Real-Time Fluorine NMR Spectroscopy for Enzyme Kinetics: Hydrolysis of N‑Trifluoroacetylglycine (TFAG) by Acylase I

The foundational Henri–Michaelis–Menten equation, derived from measurements of initial reaction rates, describes enzyme kinetics using the Michaelis–Menten constant (KM) and maximum velocity (Vmax). While traditional methods focus on initial rates, progress curve analysis, which monitors the entire reaction process, provides an alternative approach to determining kinetic parameters. This study highlights the utility of real-time quantitative 19F NMR, combined with progress curve analysis, for measuring enzyme kinetics, particularly in the hydrolysis of N-trifluoroacetylglycine (TFAG) into glycine and trifluoroacetic acid (TFA) by Acylase I. The method is further extended to examine product inhibition at higher substrate concentrations and to determine the inhibition constant with a competitive inhibitor, Butylmalonic acid. By using Pulse Length Based Concentration Determination (PULCON) to estimate concentrations independently, kinetic parameters were measured. This approach demonstrates that combining real-time quantitative 19F NMR with progress curve analysis is an effective method for conducting comprehensive kinetic studies involving fluorinated substrates.