Time‐resolved solid‐state REDOR NMR studies of UDP N‐acetylglucosamine enolpyruvyl transferase

FEBS Letters

Amrhein, Nikolaus²  Li, Yan¹  Krekel, Florian²  Ramilo, Cecilia A.¹  Evans, Jeremy N.S.¹ 

[1] Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, USA;Institut für Pflanzenwissenschaften, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland

关键词: Enolpyruvyl transferase;    Enzyme active site;    Enzyme mechanism;    Rotational echo double resonance;    Time-resolved solid-state NMR;    Uridine diphosphate N-acetylglucosamine enolpyruvyl transferase;    DTT;    dithiothreitol;    EPSP;    5-enolpyruvylshikimate-3-phosphate;    EPT;    enolpyruvyl transferase;    MOPS;    3-[N-morpholino]propanesulfonic acid;    PEP;    phosphoenolpyruvate;    REDOR;    rotational echo double resonance;    UDP-NAG;    uridine diphosphate N-acetylglucosamine;   

DOI  :  10.1016/0014-5793(95)01338-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The new method of time-resolved solid-state rotational echo double resonance (REDOR) NMR spectroscopy introduced recently by this laboratory has been applied to the enzyme uridine N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase (EPT), with the goal of probing the interactions between reactive species and their enzyme active site. The approach has been used in a qualitative fashion with the enzyme-inhibitor and enzyme-intermediate complexes of uniformly ¹⁵N-labeled UDP-NAG EPT, trapped under steady-state and pre-steady-state conditions. A different set of intermolecular interactions between the substrates UDP-NAG, UDP-NAG plus 3-Z-fluorophosphoenolpyruvate, covalent O-phosphothioketal, and UDP-NAG plus phosphoenolpyruvate trapped under time-resolved conditions (after 50 ms reaction time), and the EPT enzyme active site were observed, and this is contrasted to a similar study of the interactions in a related enzyme, 5-enolpyruvyl-shikimate-3-phosphate synthase.

【 授权许可】

Unknown