Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

• Published in: Biochemistry (Easton) Vol. 28; no. 11; pp. 4650 - 4655
• Main Authors: Donarski, William J, Dumas, David P, Heitmeyer, David P, Lewis, Vincent E, Raushel, Frank M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 30-05-1989
• Subjects: 550201 - Biochemistry- Tracer Techniques; Analytical, structural and metabolic biochemistry; AROMATICS; Aryldialkylphosphatase; BACTERIA; BARYONS; BASIC BIOLOGICAL SCIENCES; Binding Sites; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; CHEMICAL REACTIONS; DECOMPOSITION; DEUTERIUM; ELEMENTARY PARTICLES; ENZYMES; Enzymes and enzyme inhibitors; ESTERASES; FERMIONS; Fundamental and applied biological sciences. Psychology; HADRONS; HYDROGEN ISOTOPES; Hydrogen-Ion Concentration; HYDROLASES; HYDROLYSIS; HYDROXY COMPOUNDS; ISOTOPE EFFECTS; ISOTOPES; KINETICS; LIGHT NUCLEI; LYSIS; MICROORGANISMS; NUCLEI; NUCLEONS; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; Paraoxon - metabolism; Parathion - metabolism; PHENOLS; Phenols - metabolism; PHOSPHATASES; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - isolation & purification; phosphotriesterase; PROTONS; PSEUDOMONAS; Pseudomonas - enzymology; REACTION KINETICS; Solvents; SOLVOLYSIS; SPECIFICITY; STABLE ISOTOPES; Structure-Activity Relationship; STRUCTURE-ACTIVITY RELATIONSHIPS; Substrate Specificity; SUBSTRATES; Pseudomonadales; Enzymatic activity; Pseudomonas diminuta; Structure activity relation; Enzyme; Substrate specificity; Bacteria; Pseudomonadaceae; Kinetic parameter
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00437a021

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Brønsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.