A kinetic study of the irreversible inhibition of an enzyme measured in the presence of coupled enzymes. Fluorescein isothiocyanate as inhibitor of the adenosinetriphosphatase activity from sarcoplasmic reticulum

Abstract

A systematic procedure for the kinetic study of irreversible inhibition, when the enzymatic activity is measured in the presence of a coupled enzyme system, has been developed and analyzed. Simultaneous variation of the enzyme and inhibitor concentrations, maintaining a constant ratio between them, is recommended. The methodology is established to estimate the kinetic constants corresponding to the irreversible inhibitor. This approach is illustrated by the study of the inhibition of fluorescein isothiocyanate on the Ca 2+-ATPase activity from sarcoplasmic reticulum measured in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. Treatment of the experimental data has been carried out by non-linear regression.