MODIFICATION OF HUMAN PLACENTAL ALKALINE-PHOSPHATASE BY PERIODATE-OXIDIZED 1,N6-ETHENOADENOSINE MONOPHOSPHATE

Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon-AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon-AMP-dial). At room temperature-epsilon-AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A-epsilon-Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon-AMP-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 epsilon-AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A-epsilon-Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon-AMP-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 phosphatase.