Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta.

D P Dumas,F M Raushel

doi:10.1016/s0021-9258(18)45766-3

Abstract

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.

Highlights

The pH rate profile for the hydrolysis of diethyl-pnitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity
The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(Z-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity
The pKi”act for diethyl pyrocarbonate modification is 6.1 i 0.1 at 25 ‘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

Summary

PROCEDURES

Materials-The phosphotriesterase from P. diminuta was purified to homogeneity from a baculovirus expression system as described previously (Dumas et al, 1989). Enzyme Assays-The activity of phosphotriesterase was determined spectrophotometrically using a Gilford Model 260 spectrophotometer in 150 mM 2-N-(cyclohexylamino)ethanesulfonic acid (CHES), pH 9.0, buffer containing 1.0 mM diethyl-p-nitrophenyl phosphate (paraoxon) by monitoring the increase in absorbance at 400 nm resulting from the release of p-nitrophenol Reagents-The inactivation of phosphotriesterase by chemical modification reagents was explored by the addition of a large excess of reagent to an enzyme solution and incubated at 25 “C. The inactivation of phosphotriesterase by modification of lysyl residues was investigated by the addition of 100 mM pyridoxal to a 4.0 nM enzyme solution in.

RESULTS

DISCUSSION

50 PM DTNB

D P Dumas and F M Raushel