Studies on the active center of alkaline phosphatase of E. coli.

Alkaline phosphatase of E. coli is a zinc metalloenzyme,' and the activity of the enzyme can be reversibly abolished and restored by the removal and addition of zinc ions at the active site.3The metal has also been thought to stabilize the struc- ture of the enzyme.4When assayed in 1 M Tris buffer, pH 8.0, the activity, measured by formation of p-nitrophenol from p-nitrophenylphosphate, is significantly higher than in veronal.2This increased activity is due to phosphotransferase activity of the enzyme, with Tris acting as the acceptor, as demonstrated by Wilson, Dayan, and Cyr.5 A number of di-, poly-, and amino hydroxy compounds also ac- cept phosphate: 0-phosphorylethanolamine has been identified as the product with ethanolamine.5We have recently performed inorganic and organic modifications, designed to characterize both the metal and substrate binding sites of the enzyme, and found that some of these affect the hydrolase and the phosphotransferase activities differentially.This paper summarizes the results and implications of such recent experi- ments.