Direct NMR-spectroscopic determination of active-enzyme concentration by titration with a labeled inhibitor: determination of the k(cat) value of almond beta-glucosidase.
A new method for the determination of active-enzyme concentration of a glucosidase by using (13)C NMR spectroscopy is reported. The method consists of quantifying the binding between a (13)C-labelled, strong competitive inhibitor, [5-(13)C]-1-azafagomine (1), and the enzyme. The concentration of free inhibitor 1 is measured in a series of binding experiments from the intensity of its NMR signal relative to that of a reference. From a plot of the concentrations of bound vs. free inhibitor 1, the amount of specifically bound 1, that is, the amount of active sites, is determined. From this value, active-enzyme concentration and k(cat) value can be calculated.
Hansen
S U
SU
Department of Chemistry Aarhus University 8000 Aarhus C Denmark.
Plesner
I W
IW
Bols
M
M
D001667:Binding, Competitive; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006027:Glycosides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007212:Indolizines / Q000494:pharmacology*; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013997:Time Factors; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
C414749:1-azafagomine; D004791:Enzyme Inhibitors; D006027:Glycosides; D007212:Indolizines; D001617:beta-Glucosidase