[en] In the chapters dealing with enzyme reactions, the authors of all Biochemistry textbooks and of even more specialized texts consider that the characteristic parameters (kcat and Km) must be determined under initial or steady-state rate conditions. This implies the transformation of a very limited proportion of substrate (at most 10-20%) or a continuous recording of the product or substrate concentration vs. time. Both options can present practical difficulties. Is it possible to get around these very stringent conditions? Here we show that in the most favourable cases up to 70% of the substrate can be converted resulting in systematic errors on the parameters (that can easily be taken account of) if the simple Henri-Michaelis-Menten equation is utilised. Alternatively, the integrated form of the same equation directly yields excellent estimates of the same parameters. Our observations should greatly facilitate the task of researchers who study systems in which measurements of the reaction progress are painstaking or when substrate concentrations close to the detection limit must be used. The general conclusion is that it is not always absolutely necessary to determine initial or steady-state rates to obtain reliable estimations of the enzyme kinetic parameters..
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP)
Verlaine, Olivier ; Université de Liège - ULiège > Aérospatiale et Mécanique (A&M)
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Language :
English
Title :
The measurement of true initial rates is not always absolutely necessary to estimate enzyme kinetic parameters.
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