An asparagine-phenylalanine substitution accounts for catalytic differences between hGSTM3-3 and other human class mu glutathione S-transferases.
The hGSTM3 subunit, which is preferentially expressed in germ-line cells, has the greatest sequence divergence among the human mu class glutathione S-transferases. To determine a structural basis for the catalytic differences between hGSTM3-3 and other mu class enzymes, chimeric proteins were designed by modular interchange of the divergent C-terminal domains of hGSTM3 and hGSTM5 subunits. Replacement of 24 residues of the C-terminal segment of either subunit produced chimeric enzymes with catalytic properties that reflected those of the wild-type enzyme from which the C-terminus had been derived. Deletion of the tripeptide C-terminal extension found only in the hGSTM3 subunit had no effect on catalysis. The crystal structure determined for a ligand-free hGSTM3 subunit indicates that an Asn212 residue of the C-terminal domain is near a hydrophobic cluster of side chains formed in part by Ile13, Leu16, Leu114, Ile115, Tyr119, Ile211, and Trp218. Accordingly, a series of point mutations were introduced into the hGSTM3 subunit, and it was indeed determined that a Y119F mutation considerably enhanced the turnover rate of the enzyme for nucleophilic aromatic substitution reactions. A more striking effect was observed for a double mutant (Y119F/N212F) which had a k(cat)/K(m)(CDNB) value of 7.6 x 10(5) s(-)(1) M(-)(1) as compared to 4.9 x 10(3) s(-)(1) M(-)(1) for the wild-type hGSTM3-3 enzyme. The presence of a polar Asn212 in place of a Phe residue found in the cognate position of other mu class glutathione S-transferases, therefore, has a marked influence on catalysis by hGSTM3-3.
Patskovsky
Y V
YV
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Patskovska
L N
LN
Listowsky
I
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D004137:Dinitrochlorobenzene / Q000031:analogs & derivatives / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004137:Dinitrochlorobenzene; D011993:Recombinant Fusion Proteins; C022982:2-carboxy-4,6-dinitrochlorobenzene; D010649:Phenylalanine; D001216:Asparagine; D005982:Glutathione Transferase