Elucidation of the role of Arg 110 of murine leukemia virus reverse transcriptase in the catalytic mechanism: Biochemical characterization of its mutant enzymes

Kajal Chowdhury, Neerja Kaushik, Virendra N. Pandey, Mukund J. Modak

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Abstract

Based on the projected three-dimensional equivalence of conserved amino acids in the catalytic domains of DNA polymerases, we propose Arg 110 of MuLV RT to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain evidence to support this proposition and to assess the functional importance of Arg 110, we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala, and Glu. The mutant enzymes were characterized with respect to their kinetic parameters, ability to bind template-primers, and the mode of DNA synthesis. All the three substitutions at 110 position resulted in severe loss of polymerase activity without any significant effect on the RNase H function. In spite of an approximately 1000-fold reduction in k(cat) of polymerase activity with three mutant enzymes, no significant reduction in the affinities for either template primer or dNTP substrates was apparent. Mutant enzymes also did not exhibit significant sulfur elemental effect, implying that the chemical step, i.e., phosphodiester bond formation, was not defective. Examination of the mode of DNA synthesis by the mutant enzymes indicated a shift from processive to the distributive mode of synthesis. The mutants of R110 also displayed significant loss of pyrophosphorolysis activity. Furthermore, the time course of primer extension with mutant enzymes indicated severe reduction in the rates of addition of the first nucleotide and even further reduction in the addition of the second nucleotide. These results suggest that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation. Based on these results, we propose that Arg 110 of MuLV RT participates in the conformational change steps prior to and after the chemical step of polymerase reaction.

Original languageEnglish (US)
Pages (from-to)16610-16620
Number of pages11
JournalBiochemistry
Volume35
Issue number51
DOIs
StatePublished - Dec 24 1996

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Murine Leukemia Viruses
RNA-Directed DNA Polymerase
Viruses
Enzymes
Nucleotides
Catalytic DNA
Ribonuclease H
Mutagenesis
DNA Primers
DNA
DNA-Directed DNA Polymerase
Site-Directed Mutagenesis
Sulfur
Kinetic parameters
Catalytic Domain
Substitution reactions
Thermodynamic properties
Amino Acids
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Elucidation of the role of Arg 110 of murine leukemia virus reverse transcriptase in the catalytic mechanism: Biochemical characterization of its mutant enzymes",
abstract = "Based on the projected three-dimensional equivalence of conserved amino acids in the catalytic domains of DNA polymerases, we propose Arg 110 of MuLV RT to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain evidence to support this proposition and to assess the functional importance of Arg 110, we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala, and Glu. The mutant enzymes were characterized with respect to their kinetic parameters, ability to bind template-primers, and the mode of DNA synthesis. All the three substitutions at 110 position resulted in severe loss of polymerase activity without any significant effect on the RNase H function. In spite of an approximately 1000-fold reduction in k(cat) of polymerase activity with three mutant enzymes, no significant reduction in the affinities for either template primer or dNTP substrates was apparent. Mutant enzymes also did not exhibit significant sulfur elemental effect, implying that the chemical step, i.e., phosphodiester bond formation, was not defective. Examination of the mode of DNA synthesis by the mutant enzymes indicated a shift from processive to the distributive mode of synthesis. The mutants of R110 also displayed significant loss of pyrophosphorolysis activity. Furthermore, the time course of primer extension with mutant enzymes indicated severe reduction in the rates of addition of the first nucleotide and even further reduction in the addition of the second nucleotide. These results suggest that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation. Based on these results, we propose that Arg 110 of MuLV RT participates in the conformational change steps prior to and after the chemical step of polymerase reaction.",
author = "Kajal Chowdhury and Neerja Kaushik and Pandey, {Virendra N.} and Modak, {Mukund J.}",
year = "1996",
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T1 - Elucidation of the role of Arg 110 of murine leukemia virus reverse transcriptase in the catalytic mechanism

T2 - Biochemical characterization of its mutant enzymes

AU - Chowdhury, Kajal

AU - Kaushik, Neerja

AU - Pandey, Virendra N.

AU - Modak, Mukund J.

PY - 1996/12/24

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N2 - Based on the projected three-dimensional equivalence of conserved amino acids in the catalytic domains of DNA polymerases, we propose Arg 110 of MuLV RT to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain evidence to support this proposition and to assess the functional importance of Arg 110, we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala, and Glu. The mutant enzymes were characterized with respect to their kinetic parameters, ability to bind template-primers, and the mode of DNA synthesis. All the three substitutions at 110 position resulted in severe loss of polymerase activity without any significant effect on the RNase H function. In spite of an approximately 1000-fold reduction in k(cat) of polymerase activity with three mutant enzymes, no significant reduction in the affinities for either template primer or dNTP substrates was apparent. Mutant enzymes also did not exhibit significant sulfur elemental effect, implying that the chemical step, i.e., phosphodiester bond formation, was not defective. Examination of the mode of DNA synthesis by the mutant enzymes indicated a shift from processive to the distributive mode of synthesis. The mutants of R110 also displayed significant loss of pyrophosphorolysis activity. Furthermore, the time course of primer extension with mutant enzymes indicated severe reduction in the rates of addition of the first nucleotide and even further reduction in the addition of the second nucleotide. These results suggest that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation. Based on these results, we propose that Arg 110 of MuLV RT participates in the conformational change steps prior to and after the chemical step of polymerase reaction.

AB - Based on the projected three-dimensional equivalence of conserved amino acids in the catalytic domains of DNA polymerases, we propose Arg 110 of MuLV RT to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain evidence to support this proposition and to assess the functional importance of Arg 110, we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala, and Glu. The mutant enzymes were characterized with respect to their kinetic parameters, ability to bind template-primers, and the mode of DNA synthesis. All the three substitutions at 110 position resulted in severe loss of polymerase activity without any significant effect on the RNase H function. In spite of an approximately 1000-fold reduction in k(cat) of polymerase activity with three mutant enzymes, no significant reduction in the affinities for either template primer or dNTP substrates was apparent. Mutant enzymes also did not exhibit significant sulfur elemental effect, implying that the chemical step, i.e., phosphodiester bond formation, was not defective. Examination of the mode of DNA synthesis by the mutant enzymes indicated a shift from processive to the distributive mode of synthesis. The mutants of R110 also displayed significant loss of pyrophosphorolysis activity. Furthermore, the time course of primer extension with mutant enzymes indicated severe reduction in the rates of addition of the first nucleotide and even further reduction in the addition of the second nucleotide. These results suggest that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation. Based on these results, we propose that Arg 110 of MuLV RT participates in the conformational change steps prior to and after the chemical step of polymerase reaction.

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