TRANSITION-STATE STRUCTURES FOR PHOSPHORYL-TRANSFER REACTIONS OF P-NITROPHENYL PHOSPHATE

Heavy-atom isotope effects have been used to characterize the transiti on states for the aqueous hydrolysis reactions of the p-nitrophenyl ph osphate dianion and monoanion, for the reaction of the dianion in neat tert-butyl alcohol, and for the reaction catalyzed by alkaline phosph atase. The primary oxygen-18 isotope effect at the phenolic oxygen ((1 8)k(bridge)), the secondary nitrogen-15 effect ((15)k) in the nitrogen atom of the leaving group, and the secondary oxygen-18 isotope effect s in the nonbridge oxygen atoms of the phosphoryl group ((18)k(nonbrid ge)) have been measured. The isotope effects for the dianion reaction in water at 95 degrees C were (15)k = 1.0028 +/- 0.0002, (18)k(bridge) = 1.0189 +/- 0.0005, and (18)k(nonbridge) = 0.9994 +/- 0.0005. The di anion reaction in tert-butyl alcohol at 30 degrees C gave values of (1 5)k = 1.0039 +/- 0.0003, (18)k(bridge) = 1.0202 +/- 0.0008, and (18)k( nonbridge) = 0.9997 +/- 0.0016. When corrected for temperature, the re sults are very similar, indicating similar late transition state struc tures for the two reactions with little or no change in bond order bet ween the phosphorus and the nonbridge oxygen atoms. The isotope effect s on the aqueous reaction of the monoanion were (15)k = 1.0004 +/- 0.0 002, (18)k(bridge) = 1.0087 +/- 0.0003, and (18)k(nonbridge) = 1.0184 +/- 0.0005, suggesting both proton transfer and bond cleavage are rate -limiting. The isotope effects on the alkaline phosphatase reaction ar e all near unity, indicating that a nonchemical step is rate-limiting for the enzymatic reaction.