Regiospecificity of the hydrolysis of diadenosine polyphosphates catalyzed by three specific pyrophosphohydrolases.

Abstract

The different patterns of enzymatic cleavage of diadenosine polyphosphates, ApnAs, where n = 3-5, have been established by fast atom bombardment mass spectrometry, FAB MS, of the nucleotide products formed in the presence of H2(18)O. The three specific pyrophosphohydrolases, Ap3A hydrolase (EC 3.6.1.29) and (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from lupin and the (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli, manifest three different regiospecificities. The Ap3A hydrolase cleaves all four substrates tested, Ap3A, Ap4A, ApCH2ppA, and ApCHFppA, to give [18O]AMP and the corresponding unlabeled adenosine nucleotide. In each case, the enzyme cleaves at the phosphate proximate to the bound adenosine moiety. The (asymmetrical) Ap4A hydrolase cleaves both Ap4A and Ap5A to give unlabeled ATP plus [18O]AMP and [18O]ADP, respectively, and is thus seen to add water at the fourth phosphate from the bound adenosine moiety. Lastly, the (symmetrical) Ap4A hydrolase from E. coli gives beta-[18O]ADP from Ap3A, Ap4A, and Ap5A along with the unlabeled nucleotide coproducts. In addition, with Ap4A alpha S (ApspppA) as substrate for the bacterial enzyme, the products are beta-[18O]ADP and unlabeled ADP alpha S. This symmetrical enzyme is thus characterized as cleaving the polyphosphate chain at the second phosphate from the bound adenosine moiety.