Stereochemical and Kinetic Investigation of ³²P-Labeled Inorganic Phosphate Exchange Reaction Catalyzed by Primer-Independent and Primer-Dependent Polynucleotide Phosphorylase from Micrococcus luteus

The SP diastereomer of adenosine 5'-O-(1-thiodiphosphate) (ADPαS) is a substrate for the ³²P-labeled inorganic phosphate exchange reaction catalyzed by the T and I forms of polynucleotide phosphorylase. The exchange reaction occurs with retention of configuration. This exchange reaction is very slow when only ADPaS(5P) is present but is greatly activated by dinucleotide primers and ADPαS (R_P), although the latter is not a substrate for the exchange reaction. Ap(S)A(R_P) is an ~50% better activator of the exchange than the S_P diastereomer. Furthermore, high levels of the ADPαS(S_P) eliminate the activation by primers and by ADPαS (R_P). A phosphatase activity is present with the I form of the enzyme which converts ADPaS(R_P) to AMPS. This activity may be responsible for the formation of the 5'-phosphate end group for de novo polymerization or for the processivity of this reaction.