期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 117, 期 7, 页码 1970-1978出版社
WILEY
DOI: 10.1002/bit.27346
关键词
bioconjugation; DNA nanotechnology; enzyme engineering; enzyme microenvironment; organophosphates
资金
- Defense Threat Reduction Agency [HDTRA1-14-1-0045]
- Army Research Office [W911NF1410263]
Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K-M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K-M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and similar to 150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.
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