Affiliation:
1. Department of Biology, Emory University, Atlanta, Georgia 30319
2. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Abstract
ABSTRACT
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
k
cat
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
K
m
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 μM ADP, CDP, GDP, and UDP, the
K
m
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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pnp
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