Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.
The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.
D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001665:Binding Sites; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D019384:Nucleic Acid Synthesis Inhibitors; D013439:Sulfhydryl Reagents; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D012260:Ribonucleases; D005033:Ethylmaleimide