Work

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

Year: 2025

Type: article

Abstract: 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 19FNMR, 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 19FNMR with progress curve analysis is an effective method for conducting comprehensive kinetic studies involving fluorinated substrates.

Source: The Journal of Physical Chemistry B

DOI: 10.1021/acs.jpcb.5c05885

Authors: Saeed Amer, V. V. Krishnan

Institutions: California State University System, California State University, Fresno, University of California Davis Medical Center, University of California, Davis

Language: English

Topic: Advanced NMR Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: National Science Foundation, National Institutes of Health, National Institute of General Medical Sciences

Awards: METABOLOMIC ANALYSES OF HER2-POSITIVE BREAST CANCER CELLS, MRI: Acquisition of a 500 MHz NMR spectrometer with a cryoprobe for expanding interdisciplinary research and education at the Fresno State, G658012659, G9000016742