Kinetic studies on the phosphorolysis of polynucleotides by polynucleotide phosphorylase.
The kinetics of the phosphorolysis of polynucleotide (as differentiated from oligonucleotide) by polynucleotide phosphorylase of Micrococcus luteus has been investigated. Double reciprocal plots of initial velocity against either inorganic phosphate or polynucleotide concentration are linear, and furthermore, the affinity of the enzyme for either substrate is unaffected by the presence of the other. dADP, an analogue of ADP product, is a competitive inhibitor with respect to Pi and polynucleotidy. (Ap)tA-cyclic-p is a competitive inhibitor with respect to Pi. The results are almost identical with both primer-independent (Form-I) and primer-dependent (Form-T) enzymes, although the various kinetic constants differ. On the vasis of these data a rapid equilibrium random Bi Bi mechanism is proposed. The demonstration of two different inhibitor constants for dADP and the difference between the Michaelis and the inhibitor constant for polyadenylic acid in polynucleotide phosphorolysis indicate at least two binding sites for polyadenylic acid and dADP on M. luteus polynucleotide phosphorylase. Its is suggested that in the phosphorolysis of long chain polymers the second binding site permits the polynucleotide to snap right back into position after removal of I mononucleotide unit and thus leads to the observed processive degradation. A general discussion of oligonucleotide and polynucleotide phosphorolysis and the differences between Form-I and Form-T enzymes in de novo synthesis and degradation of polynucleotides is presented.
D001665:Binding Sites; D001667:Binding, Competitive; D003839:Deoxyadenosines; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008837:Micrococcus / Q000201:enzymology; D009841:Oligonucleotides; D011119:Polynucleotides; D011117:Polyribonucleotide Nucleotidyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D003839:Deoxyadenosines; D003854:Deoxyribonucleotides; D009841:Oligonucleotides; D011119:Polynucleotides; D011117:Polyribonucleotide Nucleotidyltransferase; D008274:Magnesium