Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function.
Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
Nanduri
V B
VB
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103.
Modak
M J
MJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006016:Glycolates; D008239:Lysine; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D010449:Peptide Mapping; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012965:Sodium Chloride; D013698:Templates, Genetic; D014357:Trypsin
D006016:Glycolates; C052188:4-(oxoacetyl)phenoxyacetic acid; D012965:Sodium Chloride; D012194:RNA-Directed DNA Polymerase; D014357:Trypsin; D008239:Lysine