Isolation and characterization of glyoxalase I from Escherichia coli

Abstract

Glyoxalase I (S-lactoylgluthathione methylglyoxal lyase, EC 4.4.1.5) from Escherichia coli was overexpressed using DF502/pDM7 and a purification scheme developed to allow purification of the enzyme to near homogeneity. The enzyme from this source was found to be homodimeric with a molecular weight of approximately 30 kDa. The pH optimum was determined to be greater than pH 8.0. Preliminary experiments have indicated an isoelectric point for this enzyme to be between pH 4.15-5.0. Metal-chelation studies indicate that the rate of loss of enzyme activity is buffer-dependent. Attempts to identify metal ions capable of activating E. coli glyoxalase I indicated that Ni^2+ most effectively activated the enzyme followed by Mn^2+, Co^2+, Ca^2+ and Mg^2+. Surprisingly, zinc was found not to increase the activity of the enzyme in contrast to what has been observed for several other glyoxalase I enzymes. Amino acid modification studies showed that aspartic acid and/or glutamic acid may be crucial for catalytic activity or substrate binding. The Km and Vmax for the hemimercaptal for methylglyoxal and gluthathione was found to be 69 uM and 0.753 umol/min, respectively. The enzyme was found to be unstable in several buffer systems but the presence of glycerol aided stabilization of glyoxalase I.