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

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Abstract
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.