A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

FEBS Letters

Lubkowski, Jacek²  Röhm, Klaus-Heinrich¹  Schleper, Stefan¹  Wlodawer, Alexander²  Derst, Christian¹  Palm, Gottfried J.² 

[1] Institut für Physiologische Chemie, Philipps-Universität, Marburg (Lahn, Germany;Macromolecular Structure laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702-1201, USA

关键词: Asparaginase II;    Acyl-enzyme intermediate;    Threonine amidohydrolase;    Enzymatic mechanism;    EcA;    Escherichia coli asparaginase II;    ErA;    Erwinia chrysanthemi asparaginase;    PGA;    Pseudomonas 7A glutaminaseasparaginase;    AGA;    Acinetobacter glutaminasificans glutaminaseasparaginase;    WsA;    Wolinella succinogenes asparaginase;    AHA;    l-aspartic β-hydroxamate;    MES;    2-[N-morpholino]ethanesulfonic acid;    rmsd;    root mean square deviation;   

DOI  :  10.1016/0014-5793(96)00660-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Escherichia coli asparaginase II catalyzes the hydrolysis of l-asparagine to l-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.

【 授权许可】

Unknown