Structural flexibility modulates the activity of human glutathione transferase P1-1 - Role of helix 2 flexibility in the catalytic mechanism

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the sigma-complex formation, It may be a structural transition involving the ternary complex, This event can be related to diffusion-controlled motions of protein portions as k(cat)degrees/k(cat) linearly increases by raising the relative viscosity of the solution, Similar viscosity dependence has been observed for K-m(GSH), while K-m(CDNB) is independent, No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, k(cat) and K-m(GSH) Seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity, Its viscosity dependence parallels that observed for k(cat) and K-m(GSH), thereby suggesting a possible correlation between k(cat), K-m(GSH), and diffusion-controlled motion of helix 2, The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of K,GSH, whereas a similar role on k(cat) cannot be ascertained unequivocally, The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants, Furthermore, fluorescence experiments support a structural motion of about 4 Angstrom occurring between helix 2 and helix 4 when GSH binds to the G-site.