Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase.
Human placental alkaline phosphatase (EC 3.1.3.1) was inactivated by periodate-oxidized AMP. The inactivation showed saturation kinetics and could be partially prevented by the substrate AMP or the product inhibitor inorganic phosphate. 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. Our results indicate that 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 t.l.c. 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 inorganic phosphate, but lower [32P]phosphate incorporation, than the native enzyme. These results support the conclusion that a lysine residue is involved in the phosphate-binding site of human placental alkaline phosphatase.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D005260:Female; D006801:Humans; D007700:Kinetics; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D000345:Affinity Labels; D000596:Amino Acids; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C033862:periodate-oxidized AMP; C008644:nitrophenylphosphate; D000249:Adenosine Monophosphate; D000469:Alkaline Phosphatase