Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis-Menten constant (K M) of the enzyme and V max of the reaction. The obtained K M of the reaction was 14 ± 3 g L⁻¹ (41 μM). Furthermore, inhibition by adenosine 5'-monophosphate (AMP) was evaluated, and the K M App value was determined to be 12 ± 2 g L⁻¹ (35 μM) for 7.5 and 15 μM AMP, respectively, with V max decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L⁻¹ min⁻¹. Therefore, AMP exerted a non-competitive inhibition.