Evidence for SN2(P) mechanism in the phosphorylation of alkaline phosphatase by substrates

Michaelis–Menten parameters have been measured for a series of alkyl-, aryl-, and arylamido-phosphates as substrates of alkaline phosphatase. Kinetic parameters for p-bromophenylamido-phosphate were measured over a pH-range and the results, including inhibition by inorganic phosphate indicated the amides to be normal substrates of the enzyme. Little variation in the parameter k0 was observed for all substrates; the parameter k0/Km which is a measure of the rate constant for phosphorylation of the free enzyme by free substrate showed a high selectivity to steric factor in the alkyl phosphate series. The amido-phosphates were more than ten-fold less reactive than the aryl phosphates in the k0/Km parameter and were calculated to be 60,000-fold too small for the SN1 (P) mechanism. Both these results together with arguments taken from the literature point to an SN2(P) mechanism for phosphorylation involving direct nucleophilic attack at phosphorus. The substituted arylamido-phosphates show a decreasing reactivity with electron-withdrawing substituent indicating acid catalysis. The zinc(II) necessary for phosphatase action is suggested to promote an SN2(P) mechanism at phosphorus by complexing with the substrate and neutralising the negative charge on the oxygen thus activating the phosphorus to nucleophilic attack.