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

[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.

[学科分类] 生物化学/生物物理

[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