Asp super(304) of Escherichia coli acid phosphatase is involved in leaving group protonation

Site-directed mutagenesis was used to explore the role of potential proton donors in the mechanism of the Escherichia coli acid phosphatase that is encoded by the appA gene. Asp super(304) appeared to be the only carboxylic acid residue that is conserved in the protein sequences of the high molecular weight acid phosphatases. The mutations Asp super(304) Ala and Asp super(304) Glu were introduced into appA and the corresponding proteins were overexpressed in E. coli and purified to homogeneity. Only small decreases were observed for the K sub(m) values of the substrates p-nitrophenyl phosphate, fructose 1,6-diphosphate, and tripolyphosphate. However, V sub(max) was greatly decreased, and the magnitude of effect depended markedly on substrate. Both mutant proteins exhibited significantly lower V sub(max) values with fructose 1,6-diphosphate, which possesses a much poorer leaving group than do the other two substrates. The importance of the leaving group was further tested by using a number of phenyl and alkyl phosphate derivatives as substrates. A linear correlation was observed between log V sub(max) and the pK sub(a) of the substrate leaving group for catalysis by the Asp super(304) Ala mutant enzyme. These results are consistent with partition experiments using ethylene glycol as an alternate nucleophile, which indicated that for the Asp super(304) Ala protein, the formation of a phosphoenzyme intermediate is the rate-determining step, in contrast to the situation for the wild type enzyme and the His super(303) Ala mutant. In the latter case, the rate-limiting step of the reaction is interpreted to be the breakdown of phosphoenzyme. It is concluded that Asp super(304), rather than His super(303), is involved in protonation of the substrate leaving group.