Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides.
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.
Ren
Yan-Guo
YG
Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden.
Martínez
Javier
J
Kirsebom
Leif A
LA
Virtanen
Anders
A
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005095:Exoribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005601:Framycetin / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D006860:Hydrogen Bonding; D017665:Hydroxyl Radical / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D018716:Molecular Mimicry; D009355:Neomycin / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D000900:Anti-Bacterial Agents; D004256:DNA Polymerase I; D017931:DNA Primers; D005095:Exoribonucleases; D005601:Framycetin; D006639:Histidine; D017665:Hydroxyl Radical; D007501:Iron; D008274:Magnesium; D009355:Neomycin