Modification of humn placental alkaline phosphatase by periodate-oxidized 1,N⁶-ethenoadenosine monophosphate

Oxidation of 1,N⁶-ethenoadenosine monophosphate (εAMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (εAMP-dial). At room temperature εAMP-dial was unstable and underwent β-elimination to give 4',5'-anhydro-1,N⁶-ethenoadenosine dialdehyde acetal (AεAdo-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. εAMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of εAMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (AεAdo-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) εAMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphate.