Yeast glyoxalase I is a monomeric enzyme with two active sites

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a seq uence alignment of the two enzymes, The model suggests that yeast glyoxalas e I has two active sites contained in a single polypeptide, To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignme nt, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glu tamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine a nd a double mutant containing both mutations was also constructed. Steady-s tate kinetics and metal analyses of the recombinant enzymes corroborate tha t yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different, The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replac e Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the sam e reaction.