Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis.
The primary and secondary 18O isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The 18O isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.
Caldwell
S R
SR
Department of Chemistry, Texas A&M University, College Station 77843.
Raushel
F M
FM
Weiss
P M
PM
Cleland
W W
WW
D043303:Aryldialkylphosphatase; D006868:Hydrolysis; D007554:Isotopes; D010755:Organophosphates / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010261:Paraoxon / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D007554:Isotopes; D010755:Organophosphates; D011994:Recombinant Proteins; D010758:Phosphorus; C070045:O,O-diethyl O-(4-carbamoylphenyl)phosphate; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase; D010261:Paraoxon; D010100:Oxygen