Purification and properties of the phosphotriesterase from Pseudomonas diminuta.
The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.
Dumas
D P
DP
Department of Chemistry, Texas A&M University, College Station 77843.
Caldwell
S R
SR
Wild
J R
JR
Raushel
F M
FM
D000596:Amino Acids / Q000032:analysis; D043303:Aryldialkylphosphatase; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007306:Insecticides; D007700:Kinetics; D009943:Organophosphorus Compounds; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D007306:Insecticides; D009943:Organophosphorus Compounds; D013439:Sulfhydryl Reagents; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase