Abstract
The ability of enzymes to modulate the dynamics of bound substrates and cofactors is a critical feature of catalysis, but the role of dynamics has largely been approached from the perspective of the protein. Here, we use an underappreciated NMR technique, subtesla high-resolution field-cycling 31P NMR relaxometry, to interrogate the dynamics of enzyme bound substrates and cofactors in guanosine-5′-monophosphate reductase (GMPR). These experiments reveal distinct binding modes and dynamic profiles associated with the 31P nuclei in the Michaelis complexes for the deamination and hydride transfer steps of the catalytic cycle. Importantly, the substrate is constrained and the cofactor is more dynamic in the deamination complex E·GMP·NADP+, whereas the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E·IMP·NADP+. The presence of D2O perturbed the relaxation of the 31P nuclei in E·IMP·NADP+ but not in E·GMP·NADP+, providing further evidence of distinct binding modes with different dynamic properties. dIMP and dGMP are poor substrates, and the dynamics of the cofactor complexes of dGMP/dIMP are disregulated relative to GMP/IMP. The substrate 2’-OH interacts with Asp219, and mutation of Asp219 to Ala decreases the value of Vmax by a factor of 30. Counterintuitively, loss of Asp219 makes both substrates and cofactors less dynamic. These observations suggest that the interactions between the substrate 2’-OH and Asp219 coordinate the dynamic properties of the Michaelis complexes, and these dynamics are important for progression through the catalytic cycle.