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

Abstract

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 catalysis. The mutation had a marginal effect on the k cat value determined using S- D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K m value than the wild-type enzyme. The competitive inhibitor 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 fluorescence studies with the inhibitor showed that this was due to a significantly increased off-rate for the mutant enzyme. The phenolic hydroxyl group of tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that this interaction makes a significant contribution to the binding of the active-site ligand.