Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves.
The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.
Departamento de Química-Física. Escuela Politécnica Superior Universidad de Castilla-La Mancha, Avda. España, s/n. Campus Universitario, E-02071, Albacete, Spain.
M A
MA
Moruno-Dávila
C
C
Garrido-del Solo
M
M
García-Moreno
B H
BH
Havsteen
F
F
Garcia-Sevilla
F
F
Garcia-Cánovas
R
R
Varón
D001665:Binding Sites; D001667:Binding, Competitive; D004789:Enzyme Activation; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D013379:Substrate Specificity
D004798:Enzymes