EVIDENCE FOR A (TRIOSEPHOSPHATE ISOMERASE-LIKE) CATALYTIC-LOOP NEAR THE ACTIVE-SITE OF GLYOXALASE-I

The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH3 (1), C6H5 (2), C6H4Cl (3), or C6H4Br (4). For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction. However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93. In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold. These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site. Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I (100-111) and Pseudomonas putida Glx I (93-105), that shows significant sequence homology to the ''catalytic loop'' of chicken muscle triosephosphate isomerase (TIM) (165-176). The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P. putida Glx I, Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity.