SITE-DIRECTED MUTAGENESIS OF GLUTATHIONE-S-TRANSFERASE YAYA - MAPPINGTHE 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 catalysi s. Based on the available structural information, site-directed mutage nesis was conducted to examine the function of arginine, lysine, gluta mine, and proline residues surrounding the GSH binding pocket. Arginin e mutants R13K, R15K, R20K, and R20I retained partial enzymatic activi ties, while R13I and R15I lost most of their activities. Kinetic studi es showed a marked increase in K(m) toward GSH for R15I suggesting tha t arginine 15 contributes significantly to the binding of GSH in the a ctive site of glutathione S-transferase YaYa. A drastic decrease in en zymatic activities for R13I suggested the importance of the charged gr oup of arginine 13 either in maintaining the structural integrity of t he enzyme or in serving a vital role in enzymatic function. Replacemen t of glutamine 54 and 67 with glutamic acid or asparagine resulted in decreased enzymatic activities. Moreover, an 11-, 17-, and 9-fold incr ease in K(m) values toward GSH for mutant Q54E, Q54N, and Q67N was obs erved, respectively. These results suggested that glutamine 54 and 67 also contributed significantly to the binding of GSH. Proline at posit ion 56 appears to be important for maintaining the structural integrit y 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 m utants, K45R and K45I, exhibited substantially higher catalytic effici encies toward both 1-chloro-2,4-dinitrobenzene and GSH than the wild t ype enzyme. Our data clearly show that lysine 45 is not an essential r esidue for catalysis nor for GSH binding in glutathione S-transferase YaYa.