On the Mechanism of de Novo Polymerization by Form I Polynucleotide Phosphorylase of Micrococcus luteust

John F. Marlier, S. J. Benkovic

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The diastereomers of adenosine 5'-0-(l-thiodiphosphate) (ADPαS) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADPαS(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADPαS(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-β-phosphate during de novo polymerization, leading to the observed S'-phospho-poly (A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADPαS(RP) is selectively used to form 5’ termini, whereas ADPαS(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADPβS functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the β-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADPαS vs. ADPβS or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.

Original languageEnglish (US)
Pages (from-to)2349-2356
Number of pages8
Issue number10
StatePublished - May 1982

All Science Journal Classification (ASJC) codes

  • Biochemistry


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