Polynucleotides. Partial purification and properties of a polynucleotide phosphorylase from Micrococcus lysodeikticus

Abstract

A polynucleotide phosphorylase was obtained from extracts of lysed cells of M. lysodeikticus and partially purified by fractionation with ammonium sulfate and phosphorolysis of the bacterial nucleic acid. An assay for the enzyme was developed based on the acid-insoluble character of the polynucleotide. The rate of synthesis of the polymer of adenosine monophosphate is proportional to the enzyme concentration and to the initial concentration of adenosine diphosphate (ADP) up to 10-2 [image]. The rate of synthesis of polymer follows zero-order kinetics over 80-90% of the reaction; an apparent equilibrium is reached which is in favor of the polymer to the extent of about 60%. The viscosity increment of the reaction mixture follows the synthesis of the polymer and reaches a maximum value when the net synthesis of the polymer has stopped. Subsequently there is a drop in viscosity which is not associated with any decrease in the amount of polymer formed. The reaction requires Mg. At low concentrations of Mg the rate of synthesis is proportional to Mg concentration; at high concentration the reaction is inhibited by the metal. The optimum Mg concentration is low at high pH, high at low pH, and is dependent to some degree on the particular enzyme preparation. The pH optimum depends on the Mg concentration and varies from 7 or below at high Mg concentration to above 10.5 at low Mg concentration. An apparent pK exists near 9.38, with 0.4 m[image]-MgCl2, but the value of the pK depends upon several factors. The activation energy at pH 8.1 is 11.7 kcal. The relative rates of synthesis of polymers are: cytidine 5-diphosphate,l; adenosine diphosphate, 0.53;uridine 5[image]-diphosphate, 0.11; guanosine diphosphate, 0. The enzyme is also activated by Mn, Ca and Ba, but these activation effects may be the result of their interaction with cation-binding agents in the enzyme preparation. A proposed mechanism of polymerization is suggested, based on a high affinity of the enzyme for the polymer and a low affinity for the monomer. The significance of the back reaction is considered in relation to the equilibrium of the phosphorylase-polymerase reaction. The interrelationships of the equilibrium constant and such parameters as yield of polymer, molecular weight and free energy of the reaction are discussed.