Ni 2 +-activated glyoxalase i from Escherichia coli: Substrate specificity, kinetic isotope effects and evolution within the βαβββ superfamily

Abstract

The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Ni 2 + -activated E. coli glyoxalase I substrate specificity, its thiol cofactor preference, the presence or absence of any substrate kinetic isotope effects on the enzyme mechanism, or whether glyoxalase I might catalyze additional reactions similar to those exhibited by related βαβ ββ structural superfamily members. The current investigation has shown that this two-enzyme system is capable of utilizing the thiol cofactors glutathionylspermidine and trypanothione, in addition to the known tripeptide glutathione, to convert substrate methylglyoxal to non-toxic d-lactate in the presence of Ni 2 + ion. E. coli glyoxalase I, reconstituted with either Ni 2 + or Cd 2 + , was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic isotope effect for the Ni 2 + -substituted enzyme was not detected; however, the proton transfer step was observed to be partially rate limiting for the Cd 2 + -substituted enzyme. This is the first non-Zn 2 + -activated GlxI where a metal ion-dependent kinetic isotope effect using deuterium-labelled substrate has been observed. Attempts to detect a glutathione conjugation reaction with the antibiotic fosfomycin, similar to the reaction catalyzed by the related superfamily member FosA, were unsuccessful when utilizing the E. coli glyoxalase I E56A mutein. © 2011 Elsevier Inc. All rights reserved.