Binding between thermolysin and its specific inhibitor, N-phosphoryl-L-leucyl-L-tryptophan (PLT).

The interaction between thermolysin and its specific inhibitor, PLT (N-phosphoryl-L-leucyl-L-tryptophan), has been investigated by steady-state inhibitory kinetics analysis, fluorometric titration, and the stopped-flow method. The inhibitor constant of PLT, Ki, and the dissociation constant of thermolysin(E)-PLT(I) complex, Kd, are found to be smaller by a factor of 4 to 300, depending on pH, resulting in stronger binding, than those of talopeptin and phosphoramidon, but all of them show similar pH dependence. The dependence of the apparent first-order rate constant, Kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular step followed by a slow unimolecular step, (Formula: see text). The values of K-1 (the dissociation constant of the intermediate EItr) and K-2 (the backward rate constant in the unimolecular step) are not so significantly different between PLT and talopeptin, while the K+2 (forward rate constant in the unimolecular step) value for PLT is about 14 times larger than that of talopeptin (pH 5.5). These facts suggest that the forward rate of the isomerization step, EItr----EI, is much larger in the absence of the sugar moiety of talopeptin, and hence it induces the stronger binding of PLT to thermolysin than that of talopeptin.