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 | |
| State | Published - Nov 1 2003 |
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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: Journal of Protein Chemistry, Vol. 22, No. 7-8, 01.11.2003, p. 631-642.Research output: Contribution to journal › Article
}
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 - 2003/11/1
Y1 - 2003/11/1
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 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)
UR - http://www.scopus.com/inward/record.url?scp=0346101568&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0346101568&partnerID=8YFLogxK
U2 - 10.1023/B:JOPC.0000008728.17159.dd
DO - 10.1023/B:JOPC.0000008728.17159.dd
M3 - Article
VL - 22
SP - 631
EP - 642
JO - Protein Journal
JF - Protein Journal
SN - 1572-3887
IS - 7-8
ER -