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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 28, Number 5
BKCSDE 28(5)
May 20, 2007

Title	Site-directed Mutagenesis of Arginine 13 Residue in Human Glutathione S-Transferase P1-1
Author	Jong-Uk Koh, Hyun-Young Cho, Kwang-Hoon Kong*
Keywords	Arginine 13 residue, Glutathione S-transferase, Site-directed mutagenesis
Abstract	In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. K_m ^GSH, K_m^DCNB and K_m ^EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I₅₀ values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.
Page	772 - 776
Full Text	PDF

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