Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: site-directed mutagenesis of Y7 and Y116.
Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.
Contreras-Vergara
Carmen A
CA
Aquatic Molecular Biology, Centro de Investigación en Alimentación y Desarrollo, Carretera a la Victoria Km 0.6, PO Box 1735, Hermosillo, Sonora 83000, México.
Valenzuela-Soto
Elisa M
EM
Arvizu-Flores
Aldo A
AA
Sotelo-Mundo
Rogerio R
RR
Yepiz-Plascencia
Gloria
G
D000818:Animals; D001665:Binding Sites; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014443:Tyrosine; D005982:Glutathione Transferase