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 Vmax of the reaction. The obtained K(M) of the reaction was 14 +/- 3 g L(-1) (41 microM). 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 microM) for 7.5 and 15 microM 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.
López-Sánchez
M
M
Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén, 23071 Jaén, Spain.
Ayora-Cañada
M J
MJ
Molina-Díaz
A
A
Siam
M
M
Huber
W
W
Quintás
G
G
Armenta
S
S
Lendl
B
B
D000249:Adenosine Monophosphate / Q000378:metabolism*; D004867:Equipment Design; D017022:Flow Injection Analysis / Q000295:instrumentation*; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods*
D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase; C029063:fructose-1,6-diphosphate