Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.
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.
Klumpp
Klaus
K
Roche Discovery Welwyn, Welwyn Garden City, UK. klaus.klumpp@roche.com
Hang
Julie Qi
JQ
Rajendran
Surendran
S
Yang
Yanli
Y
Derosier
Andre
A
Wong Kai In
Philippe
P
Overton
Hilary
H
Parkes
Kevin E B
KE
Cammack
Nick
N
Martin
Joseph A
JA
D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D015195:Drug Design; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology*; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D016914:Ribonuclease H / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D016914:Ribonuclease H