Catalytic Mechanism and Mechanism-Based Inhibitor of Glyoxalase I
Creators: name: Zhu, Guo-Zhang
Publisher: name: Maryland Shared Open Access Repository
Year: 2007
Subjects: Glyoxalase I
Homo sapiens
Saccharomyces cerevisia
Catalytic mechanism
Inhibitor
Chemistry, Biochemistry (0487)
Health Sciences, Pharmacology (0419)

description: Human glyoxalase I is a Zn(II)-dependent isomerase that catalyzes the conversion of methylglyoxal-glutathione thiohemiacetal to S-D-lactoylglutathione. The enzyme is a realistic target for the development of therapeutic agents against cancer and anxiety-like disorders, and is a member of the unique vicinal-oxygen chelate superfamily, in which metal ions with two or three openly accessible coordination sites participate in catalysis through direct coordination to the substrate or intermediate. It is generally accepted that catalysis by glyoxalase I involves a proton-transfer pathway via an enediol/enediolate intermediate, and that the abstraction of the thiohemiacetal proton from the substrate is the rate-limiting step of the enzyme-catalyzed reaction. To understand the roles of Zn in the catalysis before the initial proton abstraction, we have developed the first substrate analogue, butyl (S-glutathionyl)glyoxalate (1), and systematically explored its interaction with the cobalt ion of the kinetically comparable Co(II)-substituted enzyme using electronic and electron paramagnetic resonance (EPR) spectroscopy. Analogue 1 is stable to the catalytic action of the enzyme and yet retains both hydroxyl and carbonyl functionalities for potential metal coordination. When 1 binds to the cobalt enzyme, it replaces two water molecules in the coordination sphere and directly coordinates to the cobalt ion through its hydroxyl and carbonyl functionalities without affecting the ligation between Co(II) and the catalytic base Glu172. In contrast, the transition-state analogue S-(N-hydroxy-N-p-chlorophenylcarbamoyl)glutathione coordinates to cobalt as a bidentate ligand and displaces Glu172 from the coordination sphere, while the product S-D-lactoylglutathione binds to the second sphere of the cobalt ion without changing the coordination status of Glu172. Zn(II) therefore not only stabilizes the enediolate intermediate and neighboring transition states in the reaction pathway, but may also reduce the pKa of the thiohemiacetal proton through direct Zn(II) coordination to achieve facile proton transfer.; The mechanism for the selective inactivation of yeast over human glyoxalase I by S-(4-bromo-2,3-dioxobutyl)glutathione (4) was investigated using mass spectrometry-based peptide mapping and EPR spectroscopy of the Co(II)-substituted enzymes. The yeast enzyme is found to contain two functional active sites with distinct coordination environment. Compound 4 inactivates the whole enzyme by covalently modifying Glu318, a putative catalytic base and metal coordination ligand in one of the active sites, without affecting the metal ion or the putative catalytic base (Glu163) of the other active site. In contrast, 4 is a poor inactivator for the human enzyme; it does not directly coordinate to the cobalt ion in the active site or alkylate the catalytic base Glu172. We attribute the lack of alkylation of the human enzyme to a rigid glutathione-binding pocket, which cannot accommodate the relatively bulky glutathiomethyl group of 4, and thus renders the electrophilic ?-bromoacyl carbon unable to reach the nucleophilic carboxylate of Glu172. This study supports the feasibility of developing species-specific inhibitors for the glyoxalase I of pathological fungi.