Analogues of adenosine 5′-diphosphate as inhibitors of polynucleotide phosphorylase: 2′-deoxyadenosine 5′-diphosphate, adenosine 5′-methylene diphosphonate, and adenosine 5′-hypophosphate Escherichia coli polynucleotide phosphorylase (polynucleotide orthophosphate nucleotidylatransferase, EC 2.7.7.8) is inhibited by 2′-deoxy-ADP, at pH 8.6, in the presence of 0.2 mM free Mg 2+, with an inhibitor constant (competitive with regards to ADP) of 8 μM. The Michaelis constant of ADP in limited elongation with (pA) 3 as primer is 700 μM under the same conditions. Two other analogues of ADP were studied under the same conditions (pH 8.6 and 0.2 mM free Mg 2+): adenosine 5′-methylene diphosphonate has no inhibitory effect, whereas adenosine 5′-hypophosphate is highly inhibitory. Its K i is 130 μM for all the reactions tested: phosphorolysis of (pA) 3, polymerization of ADP (in the presence of primer A(pA) 97) and limited elongation of (pA) 3 in the presence of dADP. In every case the K i was calculated after extrapolation at null substrate concentration, because adenosine hypophosphate inhibition is competitive of both phosphorolysis substrates, oligonucleotide and phosphate, and of nucleoside diphosphates in the elongation reaction. It is not competitive of the primer. In contrast to dADP, adenosine 5′-hypophosphate does not show different inhibition constants for polymerization and phosphorolysis. These results are discussed starting with the assumption of two distinct sites for nucleoside diphosphates, one specific for de novo synthesis, and the other being used for primer elongation.