title: Evaluation of organophosphorus chemicals-degrading enzymes: a comparison of Escherichia coli and human cytosolic aminopeptidase P.
abstract: An enzyme capable of hydrolyzing organophosphate compounds is of biological as well as environmental significance. We evaluated the possibility of human cytosolic aminopeptidase P (hcAMPP) as an attractive bioscavenger candidate by measuring the enzymatic rates of hydrolysis for a wide variety of organophosphorus compounds. The comparison of substrate specificity exhibited by hcAMPP and E. coli aminopeptidase P (E. coli AMPP) was studied. We cloned, expressed, and purified hcAMPP from HeLa cells and AMPP from Escherichia coli. The pH-rate profiles of hcAMPP were measured in the presence of organophosphate compound 3 or 5. All of the organophosphorus compounds, 1-19, were synthesized by using the approach of phosphorus chemistry described in a previous publication. The relative activity of hcAMPP and E. coli AMPP in hydrolyzing a series of organophosphorus analogues, 1-17, was evaluated in a spectrophotometric assay by monitoring the difference of accumulation of 4-nitrophenol at 400 nm. The overall substrate preference of hcAMPP is as follows: methylphosphonates>ethylphosphonates> or =organophosphates. Interestingly, the observed enhancement in the activity of hcAMPP with methyl phosphonates, 8, 10, 12, and 13, suggests that there is particularly special about the substructure of both methyl moiety and P=O ligand, since the values of specific activity with hcAMPP for the methylphosphonates 8, 10, 12, and 13 are 2- to 73-fold greater than those for the ethylphosphonates 14-17 and the organophosphates 1-7. Similarly, in E. coli AMPP toward ethylphosphonates 14-17, the results indicate that the regions of both MeO moiety and P=O ligand may be located in the vicinity of the substrate-binding site, which have not been altered within the active site of enzyme upon mutation of Trp88, Arg153, and Arg370. These studies demonstrate that E. coli AMPP and hcAMPP display different substrate preference toward organophosphorus compounds. Evidence here, therefore, represents the first example of hcAMPP that might serve as a valuable bioscavenger candidate as E. coli AMPP due to the promise from the hydrolysis of these toxic chemicals.
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Hsu
authors.forename: Chiao-Yung
authors.initials: CY
authors.lastname: Su
authors.forename: Huang-Chi
authors.initials: HC
authors.lastname: Du
authors.forename: Shu-Chuan
authors.initials: SC
authors.lastname: Jao
authors.forename: Wen-Shan
authors.initials: WS
authors.lastname: Li
mesh_terms: D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D063065:Organophosphonates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D063065:Organophosphonates; D009943:Organophosphorus Compounds; D000626:Aminopeptidases; C531476:aminopeptidase P, E coli; C531475:cytosolic aminopeptidase P, human