Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations

A very robust and inexpensive kinetic assay for determining rates of hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase is presented. The reaction increases in rate with increase in pH. The enzyme is competitively inhibited by the reaction products, uncompetitively inhibited by L-phenylalanine, and responds to the presence of two cofactors, magnesium and zinc ions. The reaction rate increases as Mg2+ concentration is increased from 1–5 mM. With increasing Zn2+ concentration, the reaction rate is stimulated and then depressed. Experimental work on the interaction between Mg2+ and Zn2+ in the reaction is suggested for more capable students.