Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.
Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
Björnestedt
R
R
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
Stenberg
G
G
Widersten
M
M
Board
P G
PG
Sinning
I
I
Jones
T A
TA
Mannervik
B
B
D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics
D007527:Isoenzymes; D009841:Oligonucleotides; D001120:Arginine; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha