Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase

Abstract

Human placental alkaline phosphatase was inactivated by periodate-oxidized AMP. The inactivation showed saturation kinetics and could be partially prevented by the substrate AMP or the product inhibitor Pi. Oxidized AMP was itself a substrate for this enzyme, with an apparent Km of 0.67 mM. The hydrolytic products of oxidized AMP were identified as oxidized adenosine hemiacetals. Oxidized AMP was also found to be a non-competitive inhibitor with respect to p-nitrophenyl phosphate, with identical Kis and Kii values of 0.15 mM. Oxidized AMP could combine with the enzyme to form a binary complex, followed by reaction with the proximal lysyl amino group to yield a Schiff base. The latter was reduced with NaBH4 and identified by TLC. The incorporation of only 1.5 molecules of oxidized [14C]AMP per enzyme subunit resulted in a complete inactivation of the enzyme. The modified enzyme showed higher apparent Km for the substrates and higher Ki for Pi, but lower [32P]phosphate incorporation, than the native enzyme. A lysine residue is probably involved in the phosphate-binding site of human placental alkaline phosphatase.