Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta.
The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.
Dumas
D P
DP
Department of Chemistry, Texas A&M University, College Station 77843.
Raushel
F M
FM
D043303:Aryldialkylphosphatase; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010261:Paraoxon / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013816:Thermodynamics
D006639:Histidine; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase; D004047:Diethyl Pyrocarbonate; D010261:Paraoxon