PmGSTB1-1 (Proteus mirabilis glutathione S-transferase B1-1) has two tryptophan residues at positions 97 and 164 in each monomer. Structural data for this bacterial enzyme indicated that Trp97 is positioned in the helix α4, whereas Trp164 is located at the bottom of the helix α6 in the xenobiotic-binding site. To elucidate the role of the two tryptophan residues they were replaced by site-directed mutagenesis. Trp97 and Trp164 were mutated to either phenylalanine or alanine. A double mutant was also constructed. The effects of the replacement on the activity, structural properties and antibiotic-binding capacity of the enzymes were examined. On the basis of the results obtained, Trp97 does not seem to be involved in the enzyme active site and structural stabilization. In contrast, different results were achieved for Trp164 mutants. Conservative substitution of the Trp164 with phenylalanine enhanced enzyme activity 10-fold, whereas replacement with alanine enhanced enzyme activity 17-fold. Moreover, the catalytic efficiency for both GSH and 1-chloro-2,4-dinitrobenzene substrates improved. In particular, the catalytic efficiency for 1-chloro-2,4-dinitrobenzene improved for both W164F (Trp164→Phe) and W164A by factors of 7- and 22-fold respectively. These results are supported by molecular graphic analysis. In fact, W164A presented a more extensive substrate-binding pocket that could allow the substrates to be better accommodated. Furthermore, both Trp164 mutants were significantly more thermolabile than wild-type, suggesting that the substitution of this residue affects the overall stability of the enzyme. Taken together, these results indicate that Trp164 is an important residue of PmGSTB1-1 in the catalytic process as well as for protein stability.
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Research Article|
December 14 2004
Contribution of the two conserved tryptophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1
Nerino ALLOCATI;
Nerino ALLOCATI
1
*Dipartimento di Scienze Biomediche, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
1To whom correspondence should be addressed (email n.allocati@dsb.unich.it).
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Michele MASULLI;
Michele MASULLI
*Dipartimento di Scienze Biomediche, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
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Marilena PIETRACUPA;
Marilena PIETRACUPA
*Dipartimento di Scienze Biomediche, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
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Bartolo FAVALORO;
Bartolo FAVALORO
*Dipartimento di Scienze Biomediche, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
†Centro Studi per l’Invecchiamento, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
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Luca FEDERICI;
Luca FEDERICI
‡Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, U.K.
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Carmine DI ILIO
Carmine DI ILIO
*Dipartimento di Scienze Biomediche, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
†Centro Studi per l’Invecchiamento, Universita' ‘G. d'Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
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Publisher: Portland Press Ltd
Received:
May 26 2004
Revision Received:
August 12 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 37–43.
Article history
Received:
May 26 2004
Revision Received:
August 12 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Citation
Nerino ALLOCATI, Michele MASULLI, Marilena PIETRACUPA, Bartolo FAVALORO, Luca FEDERICI, Carmine DI ILIO; Contribution of the two conserved tryptophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1. Biochem J 1 January 2005; 385 (1): 37–43. doi: https://doi.org/10.1042/BJ20040890
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