Functional residues on the enzyme active site of glyoxalase I from bovine brain

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dime ric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 mu. The total of eight tryptophan residues/m olecule was estimated by using a fluorescence titration method. Low values of Stem Volmer quenching constants for the quenchers used indicated that th e tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human eryt hrocytes. The activity of bovine brain glyoxalase I was found to be particularly sens itive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for a rginine and histidine residues, respectively, A minor effect was observed b y treatment of the enzyme with other amino acid-specific reagents. A protec tive effect of the competitive inhibitor S-hexylglutathione was observed fo r all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.