The Effect of Chain Length on the Phosphorolysis of Oligonucleotides by Polynucleotide Phosphorylase

The kinetics of phosphorolysis of compounds in the series (Ap)nA (n = 2 to 8), p(Ap)nA (n = 2 to 4), and (Up)nU, (n = 3 to 6) has been studied. Km and Vmax values were obtained for all compounds. Km decreases with increasing chain length and reaches a minimum value with compounds containing 5 phosphate residues. With the shorter compounds the dependence of Km on the nucleoside residues is also apparent. Experiments with inhibitory oligonucleotides such as (Ap)nA-cyclic-p and (Ap)nA-2',3'-p confirm the results with substrates. The Vmax for all oligonucleotide substrates is greater than the Vmax for polyadenylic acid. Vmax reaches an optimum value with oligonucleotides having 5 nucleoside residues. On the basis of these results, a model for the active center of polynucleotide phosphorylase is proposed. This model has multiple subsites for the interaction of the enzyme with polynucleotides. Similar results were obtained with an enzyme form which does not require oligonucleotide primer in the polymerization reaction and an enzyme form which does require primer.