Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide

Xinyi Zhan, Cheng Keat Tan, Walter A. Scott, A. Mohsin Mian, Kathleen M. Downey, Antero G. So

Research output: Contribution to journalArticle

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Abstract

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly (rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates. These findings suggest that the RNase H activity of HIV-1 reverse transcriptase may undergo conformational changes upon interaction with RNA/DNA hybrids and that these changes are dependent on the divalent cation activator.

Original languageEnglish
Pages (from-to)1366-1372
Number of pages7
JournalBiochemistry
Volume33
Issue number6
StatePublished - Dec 1 1994
Externally publishedYes

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Ribonuclease H
Ethylmaleimide
Divalent Cations
Conformations
Poly T
Substrates
Poly A
Hydrolysis
Exonucleases
Enzymes
Ribonucleases
Human immunodeficiency virus 1 reverse transcriptase
3'-azido-3'-deoxythymidine 5'phosphate
Nucleotides
Chemical analysis
RNA
Substitution reactions
DNA
Kinetics
poly A-T

ASJC Scopus subject areas

  • Biochemistry

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Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide. / Zhan, Xinyi; Tan, Cheng Keat; Scott, Walter A.; Mohsin Mian, A.; Downey, Kathleen M.; So, Antero G.

In: Biochemistry, Vol. 33, No. 6, 01.12.1994, p. 1366-1372.

Research output: Contribution to journalArticle

Zhan, Xinyi ; Tan, Cheng Keat ; Scott, Walter A. ; Mohsin Mian, A. ; Downey, Kathleen M. ; So, Antero G. / Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide. In: Biochemistry. 1994 ; Vol. 33, No. 6. pp. 1366-1372.
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abstract = "The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly (rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates. These findings suggest that the RNase H activity of HIV-1 reverse transcriptase may undergo conformational changes upon interaction with RNA/DNA hybrids and that these changes are dependent on the divalent cation activator.",
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AU - Downey, Kathleen M.

AU - So, Antero G.

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