Reaction mechanism of glyoxalase I explored by an X-ray crystallographic analysis of the human enzyme in complex with a transition state analogue

The structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iod ophenylcarbamoyl)glutathione (HIPC-GSH) and S-p-nitrobenzyloxycarbonylgluta thione (NBC-GSH) have been determined at 2.0 and 1.72 Angstrom resolution, respectively. HIPC-GSH is a transition state analogue mimicking the enediol ate intermediate that forms along the reaction pathway of glyoxalase I. In the structure, the hydroxycarbamoyl function is directly coordinated to the active site zinc ion. In contrast, the equivalent group in the NBC-GSH com plex is approximately 6 Angstrom from the metal in a conformation that may resemble the product complex with S-D-lactoylglutathione. In this complex, two water molecules occupy the liganding positions at the zinc ion occupied by the hydroxycarbamoyl function in the enediolate analogue complex. Coord ination of the transition state analogue to the metal enables a loop to clo se down over the active site, relative to its position in the product-like structure, allowing the glycine residue of the glutathione moiety to hydrog en bond with the protein. The structure of the complex with the enediolate analogue supports an "inner sphere mechanism" in which the GSH-methylglyoxa l thiohemiacetal substrate is converted to product via a cis-enediolate int ermediate. The zinc ion is envisioned to play an electrophilic role in cata lysis by directly coordinating this intermediate. In addition, the carboxyl of Glu 172 is proposed to be displaced from the inner coordination sphere of the metal ion during substrate binding, thus allowing this group to faci litate proton transfer between the adjacent carbon atoms of the substrate. This proposal is supported by the observation that in the complex with the enediolate analogue the carboxyl group of Glu 172 is 3.3 Angstrom from the metal and is in an ideal position for reprotonation of the transition state intermediate. In contrast, Glu 172 is directly coordinated to the zinc ion in the complexes with S-benzylglutathione and with NBC-GSH.