15N-1H HSQC NMR evidence for distinct specificity of two active sites in Escherichia coli glyoxalase I.
Much remains to be elucidated concerning the selectivity mechanism of supposedly identical active sites in oligomeric proteins. Glyoxalase I (GlxI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. The E. coli GlxI is a Ni(2+)/Co(2+)-activated homodimeric protein containing two symmetric, and dually metallated active sites as characterized by X-ray structure determination. Nevertheless, kinetics and isothermal titration calorimetric (ITC) studies indicate that dimeric GlxI binds to metal ions in a ratio of 1:1 (one metal ion/one dimer) [ Clugston , S. L. , Yajima , R. , and Honek , J. F. ( 2004 ) Biochem. J. 377 , 309 - 316 ]. In the current study, we provide spectroscopic evidence for the nonequivalent metallation of GlxI by use of (15)N-(1)H HSQC NMR titration experiments. (15)N-(1)H HSQC NMR spectra reveal that the local conformations of the two active sites in homodimeric GlxI are initially asymmetric in the apo-form, resulting in functional differentiation, wherein only one active site binds to the Ni(2+) ion, and another active site is observed to be more selective for a potent inhibitor. The current results enhance our understanding of GlxI structure-function relationships and provide a potential new strategy for the development of small molecule inhibitors for this enzyme system.
Su
Zhengding
Z
Department of Chemistry, University of Waterloo, Waterloo ON N2L 3G1, Canada.
Sukdeo
Nicole
N
Honek
John F
JF
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007791:Lactoylglutathione Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017433:Protein Structure, Secondary; D011522:Protons; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D007791:Lactoylglutathione Lyase