Wang R W, Newton D J, Johnson A R, Pickett C B, Lu A Y
Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065.
J Biol Chem. 1993 Nov 15;268(32):23981-5.
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.
我们实验室之前的研究表明，天冬氨酸101在谷胱甘肽与大鼠谷胱甘肽S-转移酶YaYa的相互作用中起重要作用，而酪氨酸9直接参与催化作用。基于现有的结构信息，进行了定点诱变以研究谷胱甘肽结合口袋周围的精氨酸、赖氨酸、谷氨酰胺和脯氨酸残基的功能。精氨酸突变体R13K、R15K、R20K和R20I保留了部分酶活性，而R13I和R15I失去了大部分活性。动力学研究表明，R15I对谷胱甘肽的Km值显著增加，这表明精氨酸15对谷胱甘肽在谷胱甘肽S-转移酶YaYa活性位点的结合有显著贡献。R13I的酶活性急剧下降，表明精氨酸13的带电基团对于维持酶的结构完整性或在酶功能中发挥重要作用至关重要。用谷氨酸或天冬酰胺取代谷氨酰胺54和67导致酶活性降低。此外，分别观察到突变体Q54E、Q54N和Q67N对谷胱甘肽的Km值增加了11倍、17倍和9倍。这些结果表明，谷氨酰胺54和67对谷胱甘肽的结合也有显著贡献。56位的脯氨酸似乎对维持酶的结构完整性很重要，因为突变体P56A和P56F的活性比野生型酶低得多且稳定性极差。两个赖氨酸突变体K45R和K45I对1-氯-2,4-二硝基苯和谷胱甘肽的催化效率均显著高于野生型酶。我们的数据清楚地表明，赖氨酸45对于谷胱甘肽S-转移酶YaYa的催化作用和谷胱甘肽结合都不是必需残基。
