Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification.

Abstract

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

• Authors: Gurdip K Daffu, Patricia Lopez, Francine Katz, Michael Vinogradov, Chang-Guo Zhan, Donald W Landry, Joanne Macdonald
• Journal: Protein engineering, design & selection : PEDS (Protein Eng Des Sel) Vol. 28 Issue 11 Pg. 501-6 (Nov 2015) ISSN: 1741-0134 [Electronic] England
• PMID: 26243887 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].

Chemical References

• Fluoresceins
• Organophosphates
• fluorescein 5-maleimide
• Phosphoric Triester Hydrolases
• Cysteine
• Paraoxon

Topics

• Cysteine (chemistry, genetics, metabolism)
• Fluoresceins (chemistry, metabolism)
• Kinetics
• Models, Molecular
• Mutation
• Organophosphates (analysis, metabolism)
• Paraoxon (analysis, metabolism)
• Phosphoric Triester Hydrolases (chemistry, genetics, metabolism)
• Protein Engineering