Nucleic Acids Research, 2003, Vol. 31, No. 23 6852-6859
© 2003 Oxford University Press
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Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors
1 Roche Discovery Welwyn, Welwyn Garden City, UK and 2 Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
*To whom correspondence should be addressed at Roche Palo Alto LLC, Mail Stop S3-1, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Tel: +1 650 855 6028; Fax: +1 650 354 7554; Email: klaus.klumpp{at}roche.com
Present addresses:
Philippe Wong Kai In and Hilary Overton, OSI Pharmaceuticals, Oxford, UK
Kevin E. B. Parkes, Medivir AB, Cambridge, UK
Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose–response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose–response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.
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