Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V

R. C. Manuel, K. A. Latham, M. L. Dodson, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the α-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino- terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

Original languageEnglish (US)
Pages (from-to)2652-2661
Number of pages10
JournalJournal of Biological Chemistry
Volume270
Issue number6
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA Glycosylases
Glutamic Acid
Pyrimidine Dimers
Catalytic Domain
Mutant Proteins
Assays
Deoxyribonuclease (Pyrimidine Dimer)
Mutagenesis
Chemical modification
Enzymes
Viperidae
Site-Directed Mutagenesis
Catalyst activity
Crystal structure
Gels
X-Rays
X rays
Mutation
phage T4 endonuclease V

ASJC Scopus subject areas

  • Biochemistry

Cite this

Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V. / Manuel, R. C.; Latham, K. A.; Dodson, M. L.; Lloyd, Robert (Stephen).

In: Journal of Biological Chemistry, Vol. 270, No. 6, 1995, p. 2652-2661.

Research output: Contribution to journalArticle

Manuel, R. C. ; Latham, K. A. ; Dodson, M. L. ; Lloyd, Robert (Stephen). / Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 6. pp. 2652-2661.
@article{d8bfd0b2b7b047919398c25f7f5cbb50,
title = "Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V",
abstract = "Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the α-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino- terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.",
author = "Manuel, {R. C.} and Latham, {K. A.} and Dodson, {M. L.} and Lloyd, {Robert (Stephen)}",
year = "1995",
doi = "10.1074/jbc.270.6.2652",
language = "English (US)",
volume = "270",
pages = "2652--2661",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

TY - JOUR

T1 - Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V

AU - Manuel, R. C.

AU - Latham, K. A.

AU - Dodson, M. L.

AU - Lloyd, Robert (Stephen)

PY - 1995

Y1 - 1995

N2 - Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the α-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino- terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

AB - Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the α-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino- terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0028847687&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028847687&partnerID=8YFLogxK

U2 - 10.1074/jbc.270.6.2652

DO - 10.1074/jbc.270.6.2652

M3 - Article

C2 - 7852333

AN - SCOPUS:0028847687

VL - 270

SP - 2652

EP - 2661

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -