Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities: Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis

Robert D. Schrock, Robert (Stephen) Lloyd

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70 Citations (Scopus)

Abstract

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endo-nuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80 %. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the αNH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.

Original languageEnglish (US)
Pages (from-to)17631-17639
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number26
StatePublished - Sep 15 1991
Externally publishedYes

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Deoxyribonuclease (Pyrimidine Dimer)
Methylation
Catalysis
Catalyst activity
Pyrimidine Dimers
Peptides
Molecules
DNA Repair Enzymes
Lyases
Reducing Agents
High performance liquid chromatography
Reverse-Phase Chromatography
Formaldehyde
Staphylococcus aureus
High Pressure Liquid Chromatography
Amino Acids
Degradation
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities: Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis",
abstract = "Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endo-nuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80 {\%}. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the αNH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.",
author = "Schrock, {Robert D.} and Lloyd, {Robert (Stephen)}",
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N2 - Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endo-nuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80 %. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the αNH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.

AB - Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endo-nuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80 %. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the αNH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.

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