Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding site.
Organophosphorus hydrolase detoxifies a broad range of organophosphate pesticides and the chemical warfare agents (CWAs) sarin and VX. Previously, rational genetic engineering produced OPH variants with 30-fold enhancements in the hydrolysis of CWA and their analogs. One interesting variant (H254R) in which the histidine at position 254 was changed to an arginine showed a 4-fold increase in the hydrolysis of demetonS (VX analog), a 14-fold decrease with paraoxon (an insecticide), and a 183-fold decrease with DFP (sarin analog). The three-dimensional structure of this enzyme at 1.9A resolution with the inhibitor, diethyl 4-methylbenzylphosphonate (EBP), revealed that the inhibitor did not bind at the active site, but bound exclusively into a well-defined surface pocket 12 A away from the active site. This structural feature was accompanied by non-competitive inhibition of paraoxon hydrolysis by EBP with H254R, in contrast to the native enzyme, which showed competitive inhibition. These parallel structure-function characteristics identify a functional, allosteric site on the surface of this enzyme.
Grimsley
Janet K
JK
Biochemistry and Biophysics Department, Texas A&M University, College Station, TX 77843-2128, USA.
Calamini
Barbara
B
Wild
James R
JR
Mesecar
Andrew D
AD
D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000235:genetics; D002619:Chemical Warfare Agents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D009943:Organophosphorus Compounds / Q000737:chemistry*; D017354:Point Mutation / Q000235:genetics*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity / Q000235:genetics
D002619:Chemical Warfare Agents; D009943:Organophosphorus Compounds; D043303:Aryldialkylphosphatase