On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus.
The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-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, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-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 ADP alpha S vs. ADP beta S 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.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D001665:Binding Sites; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology*; D008970:Molecular Weight; D011061:Poly A; D011108:Polymers; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides
D011108:Polymers; D013873:Thionucleotides; D011061:Poly A; C018444:adenosine 5'-O-(1-thiodiphosphate); D000244:Adenosine Diphosphate; D011117:Polyribonucleotide Nucleotidyltransferase