*2.2.4 Spectrophotometric assay of OPH UAA variant activity*

The catalysis of P-O bond cleavage of paraoxon was monitored using a continuous spectrophotometric assay. Both *k*cat and *K*m values were measured and compared to WT OPH (**Table 2**). The 3,4-dihydroxy-L-phenylalanine substitution at H257 did not have any detectable activity. 4-amino-L-phenylalanine and 3-bromotyrosine substitutions at H257 resulted in a *K*m of 0.018 mM and 0.073 mM, respectively (**Table 2**). The 4-amino-L-phenylalanine substitution at H257 improved paraoxon binding by 4.6-fold compared to WT OPH, while 3-bromo-tyrosine substitution at H257 did not improve paraoxon binding compared to WT OPH. Catalytic rates, *k*cat, for these


#### **Table 2.**

*The kcat and Km measured for WT and mutant OPH.*

mutants were not calculated due to low protein concentration after elution from Ni-NTA column, which made protein concentration measurement impossible.

Non-histidine residue UAA substitution at D253 was not able to improve substrate binding. This aligns with our hypothesis that our selected UAAs are more suitable for histidine replacement.

All 3-methyl-histine substitutions are kinetically active (**Table 2**). The 3-methylhistine substitution at H230 resulted in the most improvement among all mutants, with a 14.4-fold increase in paraoxon binding. The 3-methyl-histine substitution at H55 was the most active mutant, with a 3.2-fold increase in paraoxon binding.

#### **2.3 Discussion**

An effective enzyme bio-engineering approach starts by the identification of key amino acid residues that, when altered, improve the activity of the targeted enzyme. Many successes have been demonstrated through the development of small molecule binding proteins [17] and redesign of enzyme binding sites to either accommodate a new substrate [18] or engineer novel catalytic sites [19, 20]. A few promising results in developing potential therapeutics have examined the applications of allosteric regulations in protein engineering [21–30].

OPH is capable of hydrolyzing a wide spectrum of OP compounds, but its application in neurotoxin degradation was limited due to insufficient substrate binding affinity. In our previous project on developing thermally stable OPH variants, we utilized an allosteric network modulation algorithm and molecular design suite "Eris" [31, 32]. Hotspots that enhanced allosteric network stability were identified and we produced dozens of OPH mutants exhibiting enhanced kinetics for paraoxon, but none of them improved substrate binding. Saturation mutagenesis done by Chen et al. also failed to tighten substrate binding [33].

In this work, we investigated the potential of using UAA substitutions to improve OPH substrate binding. OPH has a unique active site structure packed with histidine residues. These histidine residues form aromatic stacking network and H-bond with

### *Neurotoxin Decontamination DOI: http://dx.doi.org/10.5772/intechopen.110853*

substrate. Three GCE plasmids and four UAAs were tested successfully for OPH UAA expression. Among four UAA substitutions, 3,4-dihydroxy-L-phenylalanine substitution not only needed longer time to express, but also resulted in no detectable activity. The 3-bromotyrosine substitutions expressed well, with low yield after purification, but this substitution was not able to improve substrate binding. These observations eliminate both compounds from the potential UAA substitution list. The 4-amino-L-phenylalanine substitution expressed well, with a low protein yield after purification. This result makes kinetic measurement difficult. With a 4.6-fold improvement on substrate binding, further optimization on protein expression could still make 4-amino-L-phenylalanine a good UAA substitution candidate. The 3-methyl-His replacement achieved the highest protein expression level, and the 3-methyl-His replacement at H230 expressed the highest improvement on paraoxon binding. 3-methyl-His replacement at H230 was able to bring *K*m down to 5.8 μM, close to the nerve agent lethal dose. Since H230 is located at the OPH active site, the 3-methyl-His replacement also reduced the turnover rate of this mutation. Force fields for these UAAs are not currently available, so MD simulation cannot yet be used to examine UAA orientations at the active site nor any formation of H-bond after UAA substitution. The goal of this study is to demonstrate the proof-of-concept of the feasibility of using UAA substitution to stabilize the OPH active site and improve substrate binding affinity. Our results are promising and provide new insight into OPH bioengineering.
