Kinetics of reactions catalyzed by Escherichia coli polynucleotide phosphorylase. 2-deoxy-ADP as substrate and inhibitor

Abstract

A systematic study of the effect of deoxy‐ADP on the reactions catalyzed by polynucleotide phosphorylase from Escherichia coli confirmed its behavior as a monofunctional analog of ADP.The polymerization reaction is limited to the addition of one (or two at the most) residues of dAMP to a primer oligonucleotide. The Km of oligonucleotides in this reaction have been studied as a function of chain length, from (pA)2 to (pA)6, and for (pA)40. The Km and Vmax decrease with the chain length, as in the phosphorolysis reaction, but the variation is quantitatively different. (pA)2pdA is not a good substrate for the limited elongation, due to its poor Vmax, while it is a substrate for phosphorolysis.The dADP/Pi exchange occurs only in the presence of oligonucleotide. Comparison of ADP/Pi exchange and dADP/Pi exchange in the presence of (pA)2 seems to indicate an interaction between binding sites of the oligonucleotide and the nucleoside diphosphate. This interaction is larger with ADP than with dADP.The inhibitory properties of dADP in both polymerization of ADP and phosphorolysis were studied. In the absence of oligonucleotide, dADP increases the lag phase of polymerization, and also increases the concentration of oligonucleotide necessary to remove the lag phase. In the presence of saturating oligonucleotide, dADP behaves like a competitive inhibitor of ADP. Similarly, dADP is a competitive inhibitor of both phosphate and oligonucleotide in the phosphorolysis reaction. In the phosphorolysis of poly A it is only competitive with phosphate.The results presented here are consistent with a complete reversibility of polymerization and phosphorolysis. Namely, the oligonucleotide, when used in the phosphorolysis reaction, covers the nucleoside diphosphate binding site. The importance of the 2′OH in the different positions of the substrates is discussed.