Kinetics of Polymerization and Phosphorolysis Reactions of Escherichia coli Polynucleotide Phosphorylase

The kinetic constants of oligo and poly adenylic acids in the phosphorolysis reaction catalyzed by Escherichia coli B polynucleotide phosphorylase, were studied as a function of chain length. Michaelis constants for oligo As (chain length 3 to 6) decrease with chain length from 210 μM to 50 μM. The presence of a 5′ phosphate residue affects the K m values of the tri‐ and tetra‐nucleotides only. For long polymers, the K m is much smaller (of the order of 10 nM polymer), and the transition length to this very low K m seems to be larger than 10 and smaller than 40. Inhibition by 3′ phosphate terminated oligo A of (pA) 3 phosphorolysis is competitive. The K i is very close to the K m of the 3'OH terminated polymer of the same chain length, at least up to a chain length of 7. Addition of 3′ phosphate terminated polymers (chain length higher than 20) results in a limited amount of inhibition (fully competitive). Inhibition by (Ap) 4 of poly A phosphorolysis is mixed at the concentrations of poly A tested. Inhibition of poly A phosphorolysis by long 3′‐phosphate terminated poly A is time‐dependent, and the K i values measured are smaller than the K m values of the substrate of the same size. A working hypothesis is presented to explain those results, whereby polynucleotide phosphorylase has two groups of binding subsites where long polymers are bound during phosphorolysis. The phosphorolysis reaction itself occurs in the first region where the 3′OH end of the polymer is bound with a short residence time, while the bulk of the polymer binds to the second region with a long residence time. The second region plays no part in the phosphorolysis of oligonucleotides.