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.

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“…Active site engineering has improved reaction kinetics, with some enzymes reaching the estimated minimum second‐order rate constant for effective scavenging of 1 × 10 7 M −1 ·min −1 (Gupta et al, 2011). PEGylation has produced PTE conjugates with multi‐day circulation in animal models, but this can come at the cost of reduced kinetics (Efremenko et al, 2017; Daffu et al, 2015). PTE‐polymer conjugates also increase in vivo stability, in some cases with the only minimal kinetic penalty (Ilyushin et al, 2013; Novikov, Grimsley, Kern, Wild, & Wales, 2010; P. Zhang et al, 2019).…”