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 language | English |
|---|---|
| Pages (from-to) | 358-367 |
| Number of pages | 10 |
| Journal | BBA - Protein Structure |
| Volume | 534 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 21 Jun 1978 |
| Externally published | Yes |
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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 journal › Article
}
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.
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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
AN - SCOPUS:0017902480
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 -