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 tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.
Dragani
B
B
Dipartimento di Scienze Biomediche, Università "G. D'Annunzio", Via dei Vestini 31, Chieti, 66100, Italy.
Cocco
R
R
Ridderström
M
M
Stenberg
G
G
Mannervik
B
B
Aceto
A
A
D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D000099301:Hydroxyacylglutathione Hydrolase
D013869:Thiolester Hydrolases; D014364:Tryptophan; D000099301:Hydroxyacylglutathione Hydrolase