Arylamine N-acetyltransferases: Covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene

Zhijun Guo, Gregory M. Vath, Carston R Wagner, Patrick E. Hanna

Research output: Contribution to journalArticle

Abstract

Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide- inactivated NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.

Original languageEnglish (US)
Pages (from-to)631-642
Number of pages12
JournalProtein Journal
Volume22
Issue number7-8
StatePublished - Dec 1 2003

Fingerprint

Arylamine N-Acetyltransferase
Proteolysis
Aniline
Cricetinae
Peptides
Derivatives
Proteins
Pepsin A
Labels
Molecules
Kinetics
Affinity Labels
2-Acetylaminofluorene
Alkylating Agents
Hydrophobic and Hydrophilic Interactions
Digestion
Catalytic Domain
bromoacetanilide
aniline
2-aminofluorene

Keywords

  • 2-(Acetylamino)fluorene
  • Acetanilide
  • Affinity labeling
  • Mass spectrometry (MS)
  • N-acetyltransferases
  • Tandem mass spectrometry (MS/MS)

Cite this

Arylamine N-acetyltransferases : Covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene. / Guo, Zhijun; Vath, Gregory M.; Wagner, Carston R; Hanna, Patrick E.

In: Protein Journal, Vol. 22, No. 7-8, 01.12.2003, p. 631-642.

Research output: Contribution to journalArticle

@article{30cab4532f4249828ebc783b2d1bebfb,
title = "Arylamine N-acetyltransferases: Covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene",
abstract = "Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide- inactivated NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.",
keywords = "2-(Acetylamino)fluorene, Acetanilide, Affinity labeling, Mass spectrometry (MS), N-acetyltransferases, Tandem mass spectrometry (MS/MS)",
author = "Zhijun Guo and Vath, {Gregory M.} and Wagner, {Carston R} and Hanna, {Patrick E.}",
year = "2003",
month = "12",
day = "1",
language = "English (US)",
volume = "22",
pages = "631--642",
journal = "Protein Journal",
issn = "1572-3887",
publisher = "Springer New York",
number = "7-8",

}

TY - JOUR

T1 - Arylamine N-acetyltransferases

T2 - Covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene

AU - Guo, Zhijun

AU - Vath, Gregory M.

AU - Wagner, Carston R

AU - Hanna, Patrick E.

PY - 2003/12/1

Y1 - 2003/12/1

N2 - Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide- inactivated NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.

AB - Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide- inactivated NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.

KW - 2-(Acetylamino)fluorene

KW - Acetanilide

KW - Affinity labeling

KW - Mass spectrometry (MS)

KW - N-acetyltransferases

KW - Tandem mass spectrometry (MS/MS)

UR - http://www.scopus.com/inward/record.url?scp=55449125724&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=55449125724&partnerID=8YFLogxK

M3 - Article

VL - 22

SP - 631

EP - 642

JO - Protein Journal

JF - Protein Journal

SN - 1572-3887

IS - 7-8

ER -