Structure-function studies on a bacterial metalloisomerase, Escherichia coli glyoxalase I

Abstract

The glyoxalase system removes cytotoxic a-ketoaldehydes from the cell. The first enzyme in this system, glyoxalase I (GlxI), is a metalloenzyme which catalyzes the isomerization of the nonenzymatically formed hemiacetal of methylglyoxal and gluatathione to S-D-lactoylgluthathione. Our investigations have focused on the bacterial GlxI enzyme isolate from Escherichia coli. We have previously demonstrated that E. coli GlcI is a homodimeric protein, which is maximally active in the prescence of Ni^2+ and shows no activity with Zn^2+. This is in marked contrast to GlxI from Homo sapiens, Saccharomyces cerevisiae, and Pseudomonas putilda that are active with Zn^2+. To extend our knowledge of glyoxalase I and the factors affecting this unexpected metal activation in E. coli GlxI, numerous kinetic analyses, structural studies, and sequence comparisons have been performed.

Examination of the kinetic parameters for E. coli GlxI indicated that the Km remains relatively constant in the presence of several catalytic metal ions. However, the activity of the enzyme is significantly altered. Maximal activity is observed in the Ni^2+-reconstituted GlxI enzyme, with decreasing activity seen with the following metals; Co^2+, Mn^2+, Ge^2+, and Cd^2+. No activity was observed in the presence of Zn^2+, Cu^2+, Mg^2+, or Ca^2+. Metal analyses and isothermal titration calorimetry (ITC) were utilized to determine that the enzyme binds one mole of metal per mole of dimeric enzyme, including Zn^2+ and Cu^2+ which produce an inactive enzyme. The ITC analyses also indicated that the metal ions are very tightly bound to the enzyme with association constants (Ka) greater than 10^7-10^8 M^-1, with the exception of Mn^2+ which has a Ka of approximately 10^6 M^-1. Metal competition studies suggested that exchange of the metal ion can occur, the rate of which is dependent upon the concentration, incubation temperature, and nature of the competing metal ions. Differential scanning calorimetry indicated that the metal-bound enzyme is significantly more stable than the apoenzyme, with the melting temperatures increasing 7-21*C in the presence of a metal ion.