Kinetics of polymerization and phosphorolysis reactions of Escherichia coli polynucleotide phosphorylase. Evidence for multiple binding of polynucleotide in phosphorolysis.

Abstract

Polymerization of nucleoside diphosphates by polynucleotide phosphorylase from Escherichia coli B normally proceeds with a lag phase (exponential phase) which can be abolished by very low concentrations of 3′-OH oligonucleotides. The influence of several factors, including the nature of the oligonucleotide and magnesium concentration, on the concentration required for half activation, i. e. abolition of the lag phase, was studied; the values varied from 1 μM to < 10 nM, depending on the oligonucleotide. Evidence is presented which indicates that, even though the oligonucleotides are rapidly incorporated into the newly formed polymer at concentrations < 1 μM, de novo synthesis is activated to the same extent as the overall reaction. Oligonucleotides activate arsenolysis of ADP at the same level as polymerization. Activation by 3′-phosphate terminated oligonucleotides requires about 100 times higher concentrations and may be a result of their contamination by dephosphorylated oligonucleotides. A kinetic scheme for the polymerization reaction is presented. The essential factor in the strong binding of polymer, either added or formed directly on the enzyme, at multiple subsites, as a necessary condition for maintaining conformation of a polynucleotide phosphorylase active in de novo synthesis of polymer. Michaelis constants in the phosphorolysis reaction of oligonucleotides (pA)3 and (pA)4 are 250 and 90 μM, respectively, while those for poly A and poly U are 2 μM (nucleotide units) and for inorganic phosphate, Km < 0,7 mM. Michaelis constants of the oligonucleotides in the phosphorolysis reaction are about three orders of magnitude larger than the apparent activator constants. The temperature coefficients of polymerization and phosphorolysis between 0.4° and 37° were the same; the rates varied 25-fold over this temperature range.