Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two t ryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp19 9) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all kn own glyoxalase II sequences as well as in metal-dependent p-lactamase and a rylsulfatase. Site-directed mutagenesis has been used to construct single-t ryptophan mutants in order to characterize better the guanidine-induced unf olding intermediates. The denaturation at equilibrium of wild-type glyoxala se II, as followed by activity, intrinsic fluorescence and CD, is multiphas ic, suggesting that different regions of varying structural stability chara cterize the native structure of glyoxalase II. At intermediate denaturant c oncentration (1.2 M guanidine) a molten globule state is attained. The reac tivation of the denatured wild-type enzyme occurs only in the presence of Z n(II) ions. The results show that Zn(II) is essential for the maintenance o f the native structure of glyoxalase II and that its binding to the apoenzy me occurs during an essential step of refolding. The comparison of unfoldin g fluorescence transitions of single-trypthophan mutants with that of wildt ype enzyme indicates that the strictly conserved "zinc binding motif" is lo cated in a flexible region of the active site in which Zn(II) participates in catalysis. (C) 1999 Academic Press.