The substrate specificity of procaryotic and eucaryotic AppppA-degrading enzymes was investigated with phosphonate analogues of diadenosine 5’,5”’-P1,P4-tetraphosphate (AppppA). App(CH2)ppA (I), App(CHBr)ppA (II), and Appp(CH2)pA (III), but not Ap(CH2)pp(CH2)pA (IV), are substrates for lupin AppppA hydrolase (EC 18.104.22.168) and phosphodiesterase I (EC 22.214.171.124). None of the four analogues is hydrolyzed by bacterial AppppA hydrolase (EC 126.96.36.199), and only analogue III is degraded by yeast AppppA phosphorylase (EC 188.8.131.52). The analogues are competitive inhibitors of all four enzymes. The affinity of analogue IV is 3-40-fold lower than that of analogues I—III for all four enzymes. Introduction of one methylene (as in I and III) [or bromomethylene (as in II)] group into AppppA results in a 3-15-fold increase of its affinity for lupin and Escherichia coli AppppA hydrolases. The same modifications only negligibly (10-30%) affect its affinity for yeast AppppA phosphorylase and decrease its affinity for lupin phosphodiesterase I about 2.5-fold. The data provide further evidence for the heterogeneity among catalytic sites of all four AppppA-degrading enzymes.
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