Directed evolution studies were done with PTE for the enhancement of hydrolysis of both sarin and soman analogs. Particular attention was focused on the toxic SpRc and SpSc isomers of the soman analog, and the Sp isomer of the sarin analog. Double substitution libraries yielded several mutants that had enhanced activity for the substrates. Among them was the double mutant, H257Y-L303T, which displayed a 462-fold increase in activity for hydrolysis of the most toxic SpSc isomer of the GD analog in comparison to the wild type. Several other mutants such as the triple mutants H254R-H257A-L303T and H254R-H257S-L303T had enhancements of between 150- and 200-fold, and had also displayed a different order of stereoselectivity relative to the wild type. For these mutants, the order of preferential hydrolysis was such that the SpRc isomer was preferentially hydrolyzed first. In contrast, the order of preferential hydrolysis for the wild type was that the RpRc was hydrolyzed first, followed by the RpSc, SpRc, and then the SpSc isomer. The reversal of stereoselective preference was also seen with the double substitution library members for hydrolysis of the sarin analog isomers. However, there were no significant improvements for sarin analog hydrolysis in these libraries. Among the best mutants obtained were H254G-H257W, H254G-H257R, and H257Y, all of which had catalytic efficiencies on the order of 106 M-1 s-1 for hydrolysis of the Sp isomer.
The toxicity for analogs of sarin, soman, and VX was evaluated using Hydra
attenuata as a model organism. The toxicity of each compound was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There was a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate nerve agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogs was 2 orders of magnitude more toxic than the analog for soman and four orders of magnitude more toxic than the analog for sarin.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/149 |
| Date | 30 September 2004 |
| Creators | Lum, Karin Tien |
| Contributors | Raushel, Frank M. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 3422458 bytes, 237703 bytes, electronic, application/pdf, text/plain, born digital |
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