Contribution of the two conserved tryptophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1.
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ABSTRACT: 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 a4, whereas Trp164 is located at the bottom of the helix a6 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.
SUBMITTER: Allocati N
PROVIDER: S-EPMC1134671 | biostudies-literature | 2005 Jan
REPOSITORIES: biostudies-literature
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