A topologically conserved residue in a-helix 6 of domain II of
human glutathione transferase (hGST) A1-1 was mutated to
investigate its contribution to protein stability and the unfolding
pathway. The replacement of Leu-164 with alanine (L164A) did
not impact on the functional and gross structural properties of
native hGST A1-1. The wild-type protein unfolds via a threestate
pathway in which only folded dimer and unfolded monomer
were highly populated at equilibrium; a native-like dimeric
intermediate with partially dissociated domains I and II was
detected using stopped-¯ow ¯uorescence studies [Wallace, Sluis-
Cremer and Dirr (1998) Biochemistry 37, 5320±5328]. In the
present study, urea-induced equilibrium unfolding of L164A
hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The
unfolding kinetic pathway for L164A hGST A1-1, like that for
the wild type, is biphasic, with a fast and a slow unfolding
event; the cavity-forming mutation has a substantially greater
effect on the rate of unfolding of the fast event. The equilibrium
and kinetic unfolding data for L164A hGST A1-1 suggest that a
rapid pre-equilibrium is established between the native dimer
and a dimeric intermediate before complete domain and subunit
dissociation and unfolding. It is proposed that the topologically
conserved bulky residue in a-helix 6 plays a role in specifying and
stabilizing the core of domain II and the interface of domains I
and II.