The active-site residue Tyr-175 in human glyoxalase II contributes to binding of glutathione derivatives

Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to cat alysis. The mutation had a marginal effect on the k(cat) value determined u sing S-D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K-m value than the wild-type enzyme. The competitive inhibito r S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione had a 30-fold higher K-i value towards the mutant, than that of the wild-type. Pre-equilibrium fluor escence studies with the inhibitor showed that this was due to a significan tly increased off-rate for the mutant enzyme. The phenolic hydroxyl group o f tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that thi s interaction makes a significant contribution to the binding of the active -site ligand. (C) 2000 Elsevier Science B.V. All rights reserved.