Cysteine‐25 of adenylate kinase reacts with dithiothreitol to form an adduct upon aging of the enzyme

FEBS Letters

Pan, Xian Ming¹  Han, Yang¹  Li, Xia¹ 

[1] National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, PR China

关键词: Protein aging;    Multiple native form;    Disulfide bridge;    Dithiothreitol adduct;    Conformation interconversion;    Rate-limiting step;    AK;    adenylate kinase;    GdmHCl;    guanidine hydrochloride;    DTNB;    5′;    5′-dithio-bis(2-nitrobenzoic acid);    NBD;    7-chloro-4-nitrobenzo-2-oxa-1;    3-diazole;    DTT;    DL-dithiothreitol;   

DOI  :  10.1016/S0014-5793(01)02954-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Adenylate kinase (AK) ages in solution in the presence of DL-dithiothreitol (DTT) with a gradual activity decrease. Upon dilution with 4 M guanidine hydrochloride denatured native and aged AK, both recover to the same activity as the fresh enzyme. Mass spectroscopy and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole chloride modification kinetics studies identify that the residue cysteine-25 of the enzyme reacts with DTT to form an adduct. The formation of the unusual bridging DTT adduct of AK appears to be the result of a stable DTT–protein complex. The K _M for AMP, ADP and MgATP of the DTT-modified enzyme does not differ significantly from that of the intact enzyme, whereas the secondary and tertiary structures of the enzyme change obviously. These results indicate that cysteine-25 may not be involved directly in substrate binding, but may play an important role in maintaining secondary and tertiary structures of native AK, as well as the conformation interconversion in the catalytic cycle.

【 授权许可】

Unknown