Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis.
Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.
Zang
T M
TM
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Hollman
D A
DA
Crawford
P A
PA
Crowder
M W
MW
Makaroff
C A
CA
D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D002457:Cell Extracts / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000378:metabolism*; D000099301:Hydroxyacylglutathione Hydrolase
D002457:Cell Extracts; D007501:Iron; D010940:Plant Proteins; D013869:Thiolester Hydrolases; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase