Human glyoxalase II contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme.
Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to the consensus Zn(2) site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn(1) site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady-state kinetic studies show that Glx2 containing only 1 equiv of Zn(II) is catalytically active and that the metal ion in the consensus Zn(2) site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo but that the catalytic activity is due to Zn(II) in the Zn(1) site.
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
Pattraranee
P
Limphong
Ross M
RM
McKinney
Nicole E
NE
Adams
Brian
B
Bennett
Christopher A
CA
Makaroff
Thusitha
T
Gunasekera
Michael W
MW
Crowder
D001665:Binding Sites; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D000099301:Hydroxyacylglutathione Hydrolase
D003035:Cobalt; D007501:Iron; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase