A sensitive core region in the structure of glutathione S-transferases.
A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, site-directed mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25 degrees C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater K (m) towards GSH and a differing K (m) towards CDNB. The V (max) ranged from 44.7 to 87.0 micromol/min per mg (wild-type, 44.7 micromol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu-103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit-subunit interface. Finally, the data suggest that Leu-103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability.
Wongsantichon
Jantana
J
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand.
Harnnoi
Thasaneeya
T
Ketterman
Albert J
AJ
D000818:Animals; D000852:Anopheles / Q000201:enzymology; D001483:Base Sequence; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D005982:Glutathione Transferase