Site-directed mutagenesis of glutathione S-transferase YaYa. Mapping the glutathione-binding site.
Previous studies from our laboratory have shown that aspartic acid 101 plays an important role in glutathione interaction to rat glutathione S-transferase YaYa, while tyrosine 9 is directly involved in catalysis. Based on the available structural information, site-directed mutagenesis was conducted to examine the function of arginine, lysine, glutamine, and proline residues surrounding the GSH binding pocket. Arginine mutants R13K, R15K, R20K, and R20I retained partial enzymatic activities, while R13I and R15I lost most of their activities. Kinetic studies showed a marked increase in Km toward GSH for R15I suggesting that arginine 15 contributes significantly to the binding of GSH in the active site of glutathione S-transferase YaYa. A drastic decrease in enzymatic activities for R13I suggested the importance of the charged group of arginine 13 either in maintaining the structural integrity of the enzyme or in serving a vital role in enzymatic function. Replacement of glutamine 54 and 67 with glutamic acid or asparagine resulted in decreased enzymatic activities. Moreover, an 11-, 17-, and 9-fold increase in Km values toward GSH for mutant Q54E, Q54N, and Q67N was observed, respectively. These results suggested that glutamine 54 and 67 also contributed significantly to the binding of GSH. Proline at position 56 appears to be important for maintaining the structural integrity of the enzyme since mutants P56A and P56F were much less active and extremely less stable than that of the wild type enzyme. Both lysine mutants, K45R and K45I, exhibited substantially higher catalytic efficiencies toward both 1-chloro-2,4-dinitrobenzene and GSH than the wild type enzyme. Our data clearly show that lysine 45 is not an essential residue for catalysis nor for GSH binding in glutathione S-transferase YaYa.
Wang
R W
RW
Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065.
Newton
D J
DJ
Johnson
A R
AR
Pickett
C B
CB
Lu
A Y
AY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats
D017931:DNA Primers; D006427:Hemin; D005982:Glutathione Transferase; D005978:Glutathione