CATALYTICALLY DISTINCT CONFORMATIONS OF THE RIBONUCLEASE-H OF HIV-1 REVERSE-TRANSCRIPTASE BY SUBSTRATE CLEAVAGE PATTERNS AND INHIBITION BY AZIDOTHYMIDYLATE AND N-ETHYLMALEIMIDE

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) h as been characterized with respect to inhibition by azidothymidylate ( AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using eithe r 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)/pol y(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 com petitive inhibitor with respect to the substrate in Mn2+ whereas it be haves as an uncompetitive inhibitor in Mg2+ These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2or Mn2+ is the divalent cation activator. Consistent with this suggest ion is the alteration in the mode of cleavage of the substrate upon su bstitution 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 sub strate, hydrolysis is both endonucleolytic and exonucleolytic in eithe r Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonucle ase activity of RNase H is preferentially inhibited by AZTMP. The sens itivity 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 endon ucleolytic activity predominates. These findings suggest that the RNas e 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.