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 are 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. Here we show that the presence of D2O perturbs 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 of GMPR, with disregulated dynamics relative to GMP/IMP. Substitution of residues that interact either with substrate or cofactor also compromise GMPR catalysis and the associated binding modes and dynamic profiles are no longer congruous with the requisite chemical transformations.

Dynamic characteristics of GMP reductase complexes revealed by high-resolution 31P field-cycling NMR relaxometry

256th National Meeting (2018)

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