Active-Site Mutants of Human Glutathione S-Transferase P1-1 : (Effects of the Mutations on substrate Specificity and Inhibition Characteristics)

다국어 초록 (Multilingual Abstract)

In order to gain further insight on the relationship betweeen structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatograghy on immobililzed GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activity toward cumene hydroperoxide and on the I50 values of S-(2, 4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extends to the electrophile binding.

목차 (Table of Contents)

• 요약
• Introduction
• Materials and Methods
• Materials
• Preparation of mutant enzymes
• Determination of protein concentration
• Enzyme activity
• Inhibition studies
• Electrophoresis
• Results
• Purification and PAGE of mutant enzymes
• Substrate specificity
• Inhibition studies
• Discussion
• References