Kinetics of substrate reaction during irreversible modification of enzyme activity for enzymes involving two substrates.

Kinetic equations for the substrate reaction during simultaneous irreversible inhibition of enzyme activity for enzymes involving two substrates have been derived. It has been shown that the method proposed previously (Tsou, Acta Biochim, Biophys. Sinica 5, 398-417, 1965) for the determination of the apparent inhibition rate constants in the cases of single substrate enzymes can also be used in the present situation. Moreover, the criteria proposed to distinguish between different substrate competition types and to detect the formation of a reversible enzyme-inhibitor complex prior to the irreversible inhibition step also apply. Methods for the estimation of the microscopic rate constants have been proposed and it has been shown that irreversible inhibition kinetics can be used to distinguish between different mechanisms for substrate binding sequences.