Bacterial alkaline phosphatase. Part III. Kinetic studies with substituted phenyl phosphates and structurally related inhibitors

Abstract

Michaelis–Menten parameters have been measured for a series of substituted phenyl phosphate substrates of alkaline phosphatase (from Escherichia coli) at pH 8·00, 0·1M ionic strength, 0·1M-trishydroxymethylaminomethane buffer and 25 °C. Values for k₀ are constant but k₀/K_m varies with substituent with a Hammett sensitivity of +0·43 (r= 0·975). The results are interpreted in terms of electrophilic assistance, probably by zinc(II), during phosphorylation or binding steps of the enzyme. Substituted phenyl- and benzyl-phosphonates (structural analogues of the substrates) competitively inhibit the enzyme but the inhibition constants (K_i) do not show a regular variation with substituent. Incursion of binding modes equivalent to non-productive binding of the substrate probably cause the random binding ability of these inhibitors. Amino-substituted phenylphosphonate inhibitors exhibit ‘mixed’ inhibition at pH 8. Increase of K_i for phenylphosphonic acid with concentration of trishydroxy-methylaminomethane buffer is supposed to result from chelation of the amine in the co-ordination sphere of the zinc chelate at the active site of the enzyme. Trishydroxymethylaminomethane acts not merely as an acceptor of the phosphate from the phospho-enzyme as was previously supposed.