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High‐Resolution 31P Field Cycling NMR as a Probe of Substrates Dynamics on GMP Reductase
Author(s) -
Rosenberg Masha,
Redfield Alfred,
Roberts Mary,
Hedstrom Lizbeth
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.7
Subject(s) - chemistry , protein dynamics , cofactor , ligand (biochemistry) , stereochemistry , hydride , crystallography , molecular dynamics , computational chemistry , enzyme , biochemistry , metal , receptor , organic chemistry
GMP reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. The reaction proceeds in two steps: first GMP reacts with cysteine to create a covalent thioimidate intermediate E‐XMP* and ammonia, followed by hydride transfer from NADPH to E‐XMP* creating IMP. The crystal structure of GMPR and IMP/NADPH revealed the presence of two different NADPH conformations ‐ “in” and “out”. In the “in” conformation, the NADPH and IMP poised for the hydride transfer, while the “out” conformation is proposed to be important in the deamination step. Here we measured the 31 P relaxation rates of substrates and cofactors over a large magnetic field range using high‐resolution field cycling NMR. Dipolar relaxation rates (R 1 ) of 31 P provide dynamic information about bound ligands. We show that the 31 P relaxation rates of both bound substrates and cofactor increased dramatically at very low fields. These enhanced rates arise from dipolar interactions of the ligand 31 P bound to the large protein. There are differences in the low field R 1 profiles for the 31 P of GMP compared to IMP in their respective NADP + complexes that reflect changes in ligand binding dynamics. Different 31 P relaxation profiles consistent with different conformations in E•IMP•NADP + and E•GMP•NADP + complexes that are considered to represent different steps of the GMPR reaction. These results demonstrate the feasibility of using field cycling to investigate the dynamics of protein‐bound substrates.