Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1.
The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu(65), Ser(103) and Glu(104) are in hydrogen-bonding distance of the N-terminal amino group of the gamma-glutamyl moiety of the co-substrate, GSH. Glu(65) was mutated to either aspartic acid or leucine, and Ser(103) and Glu(104) were both mutated to alanine. Glu(65) mutants (Glu(65)-->Asp and Glu(65)-->Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser(103)-->Ala and Glu(104)-->Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser(103) and Glu(104) are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu(65) is crucial for catalysis.
Allocati
Nerino
N
Dipartimento di Scienze Biomediche, Università G. D'Annunzio, Via dei Vestini 31, I-66013 Chieti, Italy. n.allocati@dsb.unich.it
Masulli
Michele
M
Casalone
Enrico
E
Santucci
Silvia
S
Favaloro
Bartolo
B
Parker
Michael W
MW
Di Ilio
Carmine
C
D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry*; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000494:pharmacology; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011513:Proteus mirabilis / Q000201:enzymology*; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D004137:Dinitrochlorobenzene; D009841:Oligonucleotides; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D005982:Glutathione Transferase; D005978:Glutathione