title: 15N-1H HSQC NMR evidence for distinct specificity of two active sites in Escherichia coli glyoxalase I.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo ON N2L 3G1, Canada.
authors.forename: Zhengding
authors.initials: Z
authors.lastname: Su
authors.forename: Nicole
authors.initials: N
authors.lastname: Sukdeo
authors.forename: John F
authors.initials: JF
authors.lastname: Honek
mesh_terms: 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
chemical_list: D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D007791:Lactoylglutathione Lyase