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 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.
Ridderström
M
M
Department of Biochemistry, Uppsala University, Biomedical Center, Uppsala, Sweden.
Jemth
P
P
Cameron
A D
AD
Mannervik
B
B
D001665:Binding Sites; D002384:Catalysis; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D000099301:Hydroxyacylglutathione Hydrolase
D005978:Glutathione; D010649:Phenylalanine; D013869:Thiolester Hydrolases; D014443:Tyrosine; D000099301:Hydroxyacylglutathione Hydrolase