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

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Abstract
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.