*2.2.1 Identify OPH UAA mutation sites with increased active site stability*

OPH used in this study is a homodimer from *Pseudomonas diminuta*. The OPH crystal structure 1HZY [1] contains 330 amino acids and two Zn(II) metal cations in Chain A. One Zn(II) coordinates with active site residues H201, H230 and two water molecules. The other Zn(II) coordinates with active site residues H55, H57, D301 and one water molecule [9]. Though OPH's natural substrate and function remains unknown, it is very effective at hydrolyzing the P∙O bond of the phosphotriester insecticide paraoxon. OPH can also hydrolyze a wide spectrum of OP compounds containing phosphotriester (P∙O), phosphonothioate (P∙S), phosphonofluoridate (P∙F), and phosphonocyanate (P∙CN) bonds [14, 15].

Many previous studies on OPH focused only on altering the active site residues to achieve catalytic efficiency and substrate specificity. In this chapter, we extended our study of the OPH active site to include nearby active site residues as well, using the novel allosteric regulation approach. We targeted the residues near OPH active sites that do not coordinate with metal cations and are not involved in the hydrolysis reaction, but can form H-bonds with active site residues. In that way, we identified D253, which forms an H-bond with H230. An interesting observation about OPH is that its active site is dominated by four histidine residues: H55, H57, H201, and H230 (**Figure 1**). H254 and H257 also form an aromatic stacking network at the active site, while H257 plays an important role in stabilizing OPH [16]. We found that histidine's aromatic structure made it a great candidate for aromatic UAA substitution. Alterations of these histidine residues with UAA were achieved for examination of a more stable substrate binding reaction.

#### **Figure 1.**

*A. Schematic diagram of OPH catalyzed hydrolysis reaction. B. OPH active site showing all four histidine residues and their interaction with Zn(II). C. Structure of UAAs used in this study.*
