The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication.
The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I beta turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between alpha helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S(0.5)CDNB = 0.43 mm). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the Km(GSH) value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I beta turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of alpha helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to alpha helix H3" of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed.
Labrou
N E
NE
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece.
Mello
L V
LV
Clonis
Y D
YD
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000378:metabolism*; D006540:Herbicides / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000378:metabolism; D021122:Protein Subunits; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014783:Viscosity; D003313:Zea mays / Q000201:enzymology*
D006540:Herbicides; D010940:Plant Proteins; D021122:Protein Subunits; D001216:Asparagine; D005982:Glutathione Transferase; D014357:Trypsin; D000409:Alanine