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The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Ni²⁺-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 βαβββ 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 Ni²⁺ ion. E. coli glyoxalase I, reconstituted with either Ni²⁺ or Cd²⁺, was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic …