Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH)

作者：Pedroso Marcelo M; Ely Fernanda; Mitic Natasa; Carpenter Margaret C; Gahan Lawrence R; Wilcox Dean E; Larrabee James L; Ollis David L; Schenk Gerhard^*

来源：Journal of Biological Inorganic Chemistry, 2014, 19(8): 1263-1275.

DOI：10.1007/s00775-014-1183-9

• Binding affinity
• Calorimetry
• Enzyme kinetics
• Magnetic circular dichroism
• Site-directed mutagenesis

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (> 90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

• 出版日期2014-12