Document 0315
 DOCN  M9440315
 TI    Catalytically distinct conformations of the ribonuclease H of HIV-1
       reverse transcriptase by substrate cleavage patterns and inhibition by
       azidothymidylate and N-ethylmaleimide.
 DT    9404
 AU    Zhan X; Tan CK; Scott WA; Mian AM; Downey KM; So AG; Department of
       Medicine, University of Miami School of Medicine,; Florida 33101.
 SO    Biochemistry. 1994 Feb 15;33(6):1366-72. Unique Identifier : AIDSLINE
       MED/94145986
 AB    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.(ABSTRACT TRUNCATED AT 250 WORDS)
 DE    Catalysis  Cations, Divalent  Ethylmaleimide/*PHARMACOLOGY
       HIV-1/ENZYMOLOGY  Kinetics  Magnesium/PHARMACOLOGY
       Manganese/PHARMACOLOGY  Poly A/METABOLISM  Poly C/METABOLISM  Poly
       G/METABOLISM  Poly T/METABOLISM  Protein Conformation  Reverse
       Transcriptase/*CHEMISTRY  Ribonuclease H, Calf Thymus/ANTAGONISTS &
       INHIB/*CHEMISTRY/  METABOLISM  Substrate Specificity  Thymine
       Nucleotides/*PHARMACOLOGY  Zidovudine/*ANALOGS &
       DERIVATIVES/PHARMACOLOGY  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).