Identification of reactive lysines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae

FEBS Letters

Cardemil, Emilio¹  Bazaes, Sergio²  Goldie, Hughes³  Jabalquinto, Ana María¹ 

[1] Departamento de Ciencias Químicas, Facultad de Química y Biologia, Universidad de Santiago de Chile, Casilla 307, Santiago-2, Chile;Departamento de Química, Universidad Metropolitana de Ciencias de la Educación, Santiago, Chile;Department of Microbiology, University of Saskatchewan, Saskatoon, Canada

关键词: Phosphoenolpyruvate carboxykinase;    Lysyl residues;    Chemical modification;    PEPCK;    phosphoenolpyruvate carboxykinase;    PLP;    pyridoxal 5′-phosphate;    HPLC;    high-performance liquid chromatography;    TFA;    trifluoracetic acid;    PTH;    phenylthiohydantoin;    OAA;    oxalacetic acid;    HEPES;    N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid];    PEP;    phosphoenolpyruvate;   

DOI  :  10.1016/0014-5793(95)00107-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5′-phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn²⁺. After digestion of the modified protein with trypsin plus protease V-8, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by gas-phase automatic Edman degradation. Lys²⁸⁶ of bacterial PEPCK and Lys²⁸⁹ of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP-dependent phosphoenolpyruvate carboxykinases described so far.

【 授权许可】

Unknown