Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates.
We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3' single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The kcat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3' stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3' stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 microM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3' stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.
Chang
Samantha A
SA
Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30319, USA.
Cozad
Madeline
M
Mackie
George A
GA
Jones
George H
GH
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000494:pharmacology*; D010957:Plasmids; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D011117:Polyribonucleotide Nucleotidyltransferase; D012260:Ribonucleases