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

doi:10.1023/B:JOPC.0000008728.17159.dd

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 journal › Article › peer-review

9 Scopus citations

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 NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI 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 language	English (US)
Pages (from-to)	631-642
Number of pages	12
Journal	Journal of Protein Chemistry
Volume	22
Issue number	7-8
DOIs	https://doi.org/10.1023/B:JOPC.0000008728.17159.dd
State	Published - Nov 2003

Bibliographical note

Funding Information:
The authors thank Dr. Thomas Krick and Dr. LeeAnn Higgins of the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for their assistance and advice. This research was supported in part by U.S. Public Health Service Grant CA55334 from the National Cancer Institute and a Development Grant in Drug Design from the Department of Medicinal Chemistry, University of Minnesota.

Keywords

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

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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 NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI 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.}",

note = "Funding Information: The authors thank Dr. Thomas Krick and Dr. LeeAnn Higgins of the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for their assistance and advice. This research was supported in part by U.S. Public Health Service Grant CA55334 from the National Cancer Institute and a Development Grant in Drug Design from the Department of Medicinal Chemistry, University of Minnesota.",

year = "2003",

month = nov,

doi = "10.1023/B:JOPC.0000008728.17159.dd",

language = "English (US)",

volume = "22",

pages = "631--642",

journal = "Protein Journal",

issn = "1572-3887",

publisher = "Springer New York",

number = "7-8",

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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.

N1 - Funding Information: The authors thank Dr. Thomas Krick and Dr. LeeAnn Higgins of the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for their assistance and advice. This research was supported in part by U.S. Public Health Service Grant CA55334 from the National Cancer Institute and a Development Grant in Drug Design from the Department of Medicinal Chemistry, University of Minnesota.

PY - 2003/11

Y1 - 2003/11

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 NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI 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 NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI 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)

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VL - 22

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EP - 642

JO - Protein Journal

JF - Protein Journal

SN - 1572-3887

IS - 7-8

ER -

Arylamine N-Acetyltransferases: Covalent Modification and Inactivation of Hamster NAT1 by Bromoacetamido Derivatives of Aniline and 2-Aminofluorene

Abstract

Bibliographical note

Keywords

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