Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a
| Autor: | Yury Patskovsky, Larysa N. Patskovska, Irving Listowsky |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Stereochemistry
Macromolecular Substances Protein Conformation Recombinant Fusion Proteins Molecular Sequence Data Crystallography X-Ray Biochemistry Polymerase Chain Reaction Catalysis chemistry.chemical_compound Nucleophilic aromatic substitution Glutaredoxin Catalytic Domain Escherichia coli Transferase Humans Histidine Enzyme kinetics Amino Acid Sequence Cloning Molecular DNA Primers Glutathione Transferase Binding Sites biology Active site Glutathione Kinetics chemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed Sequence Alignment |
| Zdroj: | Biochemistry. 38(4) |
| ISSN: | 0006-2960 |
| Popis: | Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes. There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pKa for these enzymes. Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|