Dataset Information

High Resolution ³¹P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

ABSTRACT: The dynamic interactions of enzymes and substrates underpins catalysis, yet few techniques can interrogate the dynamics of protein-bound ligands. Here we describe the use of field cycling NMR relaxometry to measure the dynamics of enzyme-bound substrates and cofactors in catalytically competent complexes of GMP reductase. These studies reveal new binding modes unanticipated by x-ray crystal structures and reaction-specific dynamic networks. Importantly, this work demonstrates that distal interactions not usually considered part of the reaction coordinate can play an active role in catalysis. The commercialization of shuttling apparatus will make field cycling relaxometry more accessible and expand its use to additional nuclei, promising more intriguing findings to come.

SUBMITTER: Roberts MF

PROVIDER: S-EPMC9009223 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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High Resolution <sup>31</sup>P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

Roberts Mary F MF Hedstrom Lizbeth L

Frontiers in molecular biosciences 20220331

The dynamic interactions of enzymes and substrates underpins catalysis, yet few techniques can interrogate the dynamics of protein-bound ligands. Here we describe the use of field cycling NMR relaxometry to measure the dynamics of enzyme-bound substrates and cofactors in catalytically competent complexes of GMP reductase. These studies reveal new binding modes unanticipated by x-ray crystal structures and reaction-specific dynamic networks. Importantly, this work demonstrates that distal interac ...[more]

PMID: 35433832

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Project description:The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Dataset Information

High Resolution ³¹P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

Publications

High Resolution <sup>31</sup>P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

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Dataset Information

High Resolution 31P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

Publications

High Resolution <sup>31</sup>P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

High Resolution ³¹P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.