The Effects of Various Divalent Cations on the Enzyme Activity of Bovine Intestinal Alkaline Phosphatase

Abstract

2+ 2+ Alkaline phosphatases have binding sites for Zn and Mg, on which enzyme activity is 2+ 2+ 2+ 2+ 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 V and max 2+ K of the reaction for each cation. Higher enzyme activities were observed for the Co and m 2+ 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 2+ 2+ 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 2+ bond. Enzyme activity appeared to be inhibited in the assay buffer with Zn. No appreciable activity was observed in the negative control containing EDTA.