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

López-Sánchez, M.; Ayora-Cañada, M. J.; Molina-Díaz, A.; Siam, M.; Huber, W.; Quintás, G.; Armenta, S.; Lendl, B.
August 2009
Analytical & Bioanalytical Chemistry;Aug2009, Vol. 394 Issue 8, p2137
Academic Journal
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 ( KM) of the enzyme and Vmax of the reaction. The obtained KM of the reaction was 14 ± 3 g L−1 (41 μM). Furthermore, inhibition by adenosine 5′-monophosphate (AMP) was evaluated, and the K value was determined to be 12 ± 2 g L−1 (35 μM) for 7.5 and 15 μM AMP, respectively, with Vmax decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L−1 min−1. Therefore, AMP exerted a non-competitive inhibition.


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