Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.
Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.
Reddy
G
G
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103-2714.
Nanduri
V B
VB
Basu
A
A
Modak
M J
MJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D007202:Indicators and Reagents; D007501:Iron / Q000494:pharmacology*; D058085:Iron Compounds; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011188:Potassium / Q000494:pharmacology*; D017680:Potassium Compounds; D011485:Protein Binding / Q000187:drug effects; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D013698:Templates, Genetic; D014357:Trypsin
D000596:Amino Acids; D007202:Indicators and Reagents; D058085:Iron Compounds; D017680:Potassium Compounds; D018894:Reverse Transcriptase Inhibitors; C044384:potassium ferrate; D007501:Iron; D012194:RNA-Directed DNA Polymerase; D014357:Trypsin; D011188:Potassium