Faculty Collaboration Database

Converting GLX2-1 into an active glyoxalase II. Biochemistry 2010 Sep 21;49(37):8228-36

Date

08/19/2010

Pubmed ID

20715794

Pubmed Central ID

PMC2939260

DOI

10.1021/bi1010865

Scopus ID

2-s2.0-77956605554 (requires institutional sign-in at Scopus site)   9 Citations

Abstract

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5-2-fold increase in k(cat) and a decrease in K(m) as compared to the values exhibited by the double mutant. In contrast, the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equiv of Zn(II), and (1)H NMR spectra of the Co(II)-substituted analogue of this enzyme strongly suggest that the introduced histidine binds to Co(II). EPR studies indicate the presence of significant amounts a dinuclear metal ion-containing center. Therefore, an active GLX2 enzyme requires both the presence of a properly positioned metal center and significant nonmetal, enzyme-substrate contacts, with tyrosine 255 being particularly important.

Author List

Limphong P, Adams NE, Rouhier MF, McKinney RM, Naylor M, Bennett B, Makaroff CA, Crowder MW

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Arabidopsis
Catalysis
Glutathione
Histidine
Humans
Kinetics
Lactoylglutathione Lyase
Magnetic Resonance Spectroscopy
Metals
Substrate Specificity
Thiolester Hydrolases