Coenzyme binding of a folding intermediate of aspartate aminotransferase detected by HPLC fluorescence measurements

FEBS Letters

Leistler, Bernd¹  Herold, Marzell¹ 

[1] Hewlett-Packard GmbH, Waldbronn, Analytical Division, PO Box 1280, W-7517 Waldbronn 2, Germany

关键词: Folding intermediate;    Aspartate aminotransferase;    HPLC;    cAAT;    aspartate aminotransferase (EC 2.6.1.1) from Escherichia coli;    DTE;    dithiocrythritol;    EDTA;    ethylenediaminetetraacetate;    GuCl;    guanidinium chloride;    HEPES;    4-(2-hydroxyethyl)piperazine-1-ethansulphonic acid;    P-domain;    excised coenzyme binding domain of cAAT;    PLP;    pyridoxal 5′-phosphate;    SEC;    size-exclusion chromatography;   

DOI  :  10.1016/0014-5793(92)81042-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Equilibrium dissociation and unfolding of dimeric aspartate aminotransferase from Escherichia coli proceeds via two compact monomeric intermediates which have similar hydrodynamic volumes but different fluorescence properties. We probed binding of the coenzyme pyridoxal 5′-phosphate to these intermediates by coupling fluorescence detection to size-exclusion HPLC. This procedure gave additionally an internal conformational probe of the unfolding transitions of the enzyme. It was shown that the first intermediate, M, is able to bind the coenzyme, whereas the second intermediate, M, is not. It is likely that M is the correctly folded monomer of the protein.

【 授权许可】

Unknown