The Effects of Various Divalent Cations on

Alkaline phosphatases have binding sites for Zn and Mg, on which enzyme activity is dependent. In this experiment, we tested for the effects of Mg, Zn, Co and Ca on the enzymatic conversion of synthetic pNPP substrate to nitrophenol by bovine intestinal alkaline phosphatase. Different pNPP concentrations were used to determine the Vmax and Km of the reaction for each cation. Higher enzyme activities were observed for the Co and Ca tests when compared with the positive control, in which no divalent cations were added, but only at low (< 1.5 mM) substrate concentrations. The apparent increase in enzyme activity could be attributed to the displacement of Zn from its binding site by Co; this possibly led to a more stable enzyme-substrate complex and a higher release rate of phosphate from the phosphoenzyme complex, after hydrolysis of the phosphomonoester bond. Enzyme activity appeared to be inhibited in the assay buffer with Zn. No appreciable activity was observed in the negative control containing EDTA.