In vitro characterization of organophosphorus compound hydrolysis by native and recombinant human prolidase.
Human prolidase is a binuclear metalloenzyme, which can potentially function as a catalytic bioscavenger for organophosphorus (OP) nerve agents. Although the biochemical properties of native prolidase purified from human erythrocytes, liver, kidney, and fibroblast cells are well known, it is very poorly characterized with regard to its OP hydrolyzing activity. Also, the high cost of purification of large quantities of native enzyme limits its use as a bioscavenger. Thus, recombinant human prolidase with similar biochemical properties to those of native enzyme would be more suitable as a catalytic bioscavenger. In this study, we established an Escherichia coli expression system, which produced a large amount of tagged human liver prolidase that was purified to over 95% purity from the soluble fraction of cell lysate by affinity chromatography on Streptavidin-agarose resin. The catalytic properties of the recombinant enzyme were compared in vitro with those of highly purified prolidase I isolated from human erythrocytes. The catalytic properties of recombinant prolidase overlap with those of the erythrocyte-derived native enzyme. Both enzymes efficiently hydrolyzed diisopropylfluorophosphate, sarin, soman, tabun and cyclosarin, but were much less efficient at hydrolyzing paraoxon and methyl paraoxon. These results suggest that human prolidase expressed in E. coli is suitable for further development as a catalytic bioscavenger for OP nerve agents.
Division of Bacterial & Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA.
Lakshmi
L
Chandrasekaran
Tatyana
T
Belinskaya
Ashima
A
Saxena
D043303:Aryldialkylphosphatase / Q000378:metabolism; D002619:Chemical Warfare Agents / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D004150:Dipeptidases / Q000235:genetics / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002619:Chemical Warfare Agents; D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase; D004150:Dipeptidases; C022449:proline dipeptidase