Biochemical Journal

Research article

Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies



GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn2+ but maximal activation with Ni2+. To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the kcat without significantly affecting the Km for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn2+, which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant (Ka)>107 M−1, with the exception of Mn2+ (Ka of the order of 106 M−1). One of the ligands to the catalytic metal, His5, was altered to glutamine, a side chain found in the Zn2+-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn2+-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.

  • glyoxalase I
  • isothermal titration calorimetry
  • kinetics
  • mutagenesis
  • nickel


  • 1 Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, U.S.A.

  • Abbreviations used: ESMS, electrospray mass spectrometry; GlxI, glyoxalase I; ICP, inductively coupled plasma; ITC, isothermal titration calorimetry.