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

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

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

Biochemistry. 2018 Mar 16;:

Authors: Rosenberg MM, Redfield AG, Roberts M, Hedstrom L

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+, while the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E•IMP•NADP+. Moreover, the binding mode of GMP in the NADP+ complex is not consistent with the currently available crystal structures of inactive complexes. 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, for substrates and cofactors in the two stages of the catalytic cycle. dIMP and dGMP are poor substrates, with values of Vmax an order of magnitude smaller than IMP and GMP. The binding mode and dynamic profile, as monitored by field cycling, of dGMP in its cofactor complex closely resembles that of IMP in the hydride transfer complex, and thus is not compatible with deamination. The binding mode of dIMP resembles that of GMP, and thus is not suitable for hydride transfer. The substrate 2'-OH interacts with Asp219 in the crystal structures, 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 in the active site. These observations suggest that rather than position the substrate and cofactor for catalysis, 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.


PMID: 29547266 [PubMed - as supplied by publisher]



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