Hydrolysis of phosphotriesters: determination of transition states in parallel reactions by heavy-atom isotope effects.
The remote label method was used to measure primary and secondary (18)O isotope effects in the alkaline hydrolysis of O,O-diethylphosphorylcholine iodide (DEPC) and the primary (18)O effect in the alkaline hydrolysis of O,O-diethyl-m-nitrobenzyl phosphate (DEmNBP). Both the leaving group of interest (choline or m-nitrobenzyl alcohol) and ethanol can be ejected during hydrolysis due to the similarity of their pK values. The heavy-atom isotope effects were measured by isotope ratio mass spectrometry. Parallel reaction and incomplete labeling corrections were made for both systems. DEPC has a primary (18)O isotope effect of 1.041 +/- 0.003 and a secondary (18)O isotope effect of 1.033 +/- 0.002. The primary (18)O isotope effect for DEmNBP was 1.052 +/- 0.003. These large effects suggest a highly associative transition state in which the nucleophile approaches very close to the phosphorus atom to eject the leaving group. The large values are also indicative of a large compression, or general movement, on the reaction coordinate.
Anderson
M A
MA
Institute for Enzyme Research and the Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Shim
H
H
Raushel
F M
FM
Cleland
W W
WW
D002794:Choline / Q000737:chemistry; D004952:Esters / Q000138:chemical synthesis / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D006868:Hydrolysis; D007553:Isotope Labeling; D007700:Kinetics; D009574:Nitro Compounds / Q000138:chemical synthesis / Q000737:chemistry; D009587:Nitrogen Isotopes; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010103:Oxygen Isotopes; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
D004952:Esters; D009574:Nitro Compounds; D009587:Nitrogen Isotopes; C437126:O,O-diethyl-m-nitrobenzyl phosphate; C437127:O,O-diethylphosphorylcholine iodide; D009943:Organophosphorus Compounds; D010103:Oxygen Isotopes; D010767:Phosphorylcholine; D000431:Ethanol; D002794:Choline