Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis.
The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.
Labrou
Nikolaos E
NE
Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, GR-11855 Athens, Greece. lambrou@aua.gr
Rigden
Daniel J
DJ
Clonis
Yannis D
YD
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D013379:Substrate Specificity / Q000235:genetics; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D010940:Plant Proteins; D005982:Glutathione Transferase