Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase |
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Authors: | M. López-Sánchez M. J. Ayora-Cañada A. Molina-Díaz M. Siam W. Huber G. Quintás S. Armenta B. Lendl |
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Affiliation: | (1) Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén, 23071 Jaén, Spain;(2) F. Hoffmann-La Roche Ltd, Discovery Research Basel, 4070 Basel, Switzerland;(3) Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164 AC, 1060 Vienna, Austria |
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Abstract: | 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 MApp 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. |
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Keywords: | Fourier transform infrared spectroscopy Sequential injection analysis Enzyme Inhibition kinetics Fructose-1,6-bisphosphatase |
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