DNP NMR of carbohydrate converting enzymes

Dissolution dynamic nuclear polarization (DNP) NMR can be used to increase the sensitivity of 13 C NMR signal by up to four orders of magnitude [1]. This allows for real time monitoring of reactions and observation of intermediates [2]. The biggest drawback of the method is the loss of polarization with T 1 relaxation, but even with this limitation, it is possible to obtain detailed reaction parameters in less than one minute. The enzyme investigated was β -galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound intermediate, however, this evidence is based on mutant of X-ray crystallography and simulations [3]. As the natural substrate lactose does not have any quaternary carbon with long T 1, the unnatural substrate o - nitrophenyl β -D-galactopyranoside was used (figure 1) as the quaternary positions have T 1 relaxations of ca. 15 s instead of <2 s.