Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoester ase with two Zn2+ ions in its active site. Sequence homology suggests a dis tant evolutionary relationship between AP and alkaline phosphodiesterase/nu cleotide pyrophosphatase, with conservation of the catalytic metal ions. Fu rthermore, many other phosphodiesterases, although not evolutionarily relat ed, have a similar active site configuration of divalent metal ions in thei r active sites. These observations led us to test whether AP could also cat alyze the hydrolysis of phosphate diesters. The results described herein de monstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps, inorganic phosph ate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase a nd phosphatase activities with the same inhibition constant: a point mutati on that weakens phosphate binding to AP correspondingly weakens phosphate i nhibition of the phosphodiesterase activity; and mutation of active site re sidues substantially reduces both the mono- and diesterase activities. AP a ccelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K-m)/k(w)], Although this rate enhancement is substantial, it is at least 10(6)-fold less than the r ate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysi s of both phosphate monoesters and phosphate diesters, However, mutation of the active site arginine to serine, R166S, decreases the monoesterase acti vity but not the diesterase activity, suggesting that the interaction of th is arginine with the nonbridging oxygen(s) of the phosphate monoester subst rate provides a substantial amount of the preferential hydrolysis of phosph ate monoesters, The observation of phosphodiesterase activity extends the p revious observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosph atases, and phosphodiesterases within the evolutionarily related AP superfa mily. The catalytic promiscuity of AP could have facilitated divergent evol ution via gene duplication by providing a selective advantage upon which na tural selection could have acted.