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Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Published online by Cambridge University Press:  07 November 2002

YAN-GUO REN
Affiliation:
Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden
JAVIER MARTÍNEZ
Affiliation:
Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden Present address: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
LEIF A. KIRSEBOM
Affiliation:
Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden
ANDERS VIRTANEN
Affiliation:
Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden
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Abstract

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.

Type
Research Article
Copyright
2002 RNA Society

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