Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene Cross-link localization

S. M. Deyev, G. A. Afanasenko, O. L. Polyanovsky

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the ε{lunate}-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 Å (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5′-phosphate in the active site.

Original languageEnglish
Pages (from-to)358-367
Number of pages10
JournalBBA - Protein Structure
Volume534
Issue number2
DOIs
Publication statusPublished - 21 Jun 1978
Externally publishedYes

Fingerprint

Cross-Linking Reagents
Apoenzymes
Aspartate Aminotransferases
Lysine
Proteolysis
Amino Acids
Pronase
Pyridoxal Phosphate
Covalent bonds
Fluorine
Cysteine
Tyrosine
Catalytic Domain
Atoms
Peptides
1,5-difluoro-2,4-dinitrobenzene
2,4-dinitrobenzene

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene Cross-link localization. / Deyev, S. M.; Afanasenko, G. A.; Polyanovsky, O. L.

In: BBA - Protein Structure, Vol. 534, No. 2, 21.06.1978, p. 358-367.

Research output: Contribution to journalArticle

@article{c7f0c73a02a24a4d8873c08963167a24,
title = "Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene Cross-link localization",
abstract = "At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the ε{lunate}-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 {\AA} (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5′-phosphate in the active site.",
author = "Deyev, {S. M.} and Afanasenko, {G. A.} and Polyanovsky, {O. L.}",
year = "1978",
month = "6",
day = "21",
doi = "10.1016/0005-2795(78)90019-3",
language = "English",
volume = "534",
pages = "358--367",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene Cross-link localization

AU - Deyev, S. M.

AU - Afanasenko, G. A.

AU - Polyanovsky, O. L.

PY - 1978/6/21

Y1 - 1978/6/21

N2 - At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the ε{lunate}-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 Å (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5′-phosphate in the active site.

AB - At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the ε{lunate}-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 Å (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5′-phosphate in the active site.

UR - http://www.scopus.com/inward/record.url?scp=0017902480&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0017902480&partnerID=8YFLogxK

U2 - 10.1016/0005-2795(78)90019-3

DO - 10.1016/0005-2795(78)90019-3

M3 - Article

VL - 534

SP - 358

EP - 367

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 2

ER -