Biochemical and Biophysical Investigations of Non-Zinc Dependent Glyoxalase I Enzymes

Abstract

The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.