Substrate and Cofactor Dynamics on Guanosine Monophosphate Reductase Probed by High Resolution Field Cycling 31P NMR Relaxometry.
Guanosine-5'-monophosphate reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. Here we investigated the structure and dynamics of enzyme-bound substrates and cofactors by measuring31P relaxation rates over a large magnetic field range using high resolution field cycling NMR relaxometry. Surprisingly, these experiments reveal differences in the low field relaxation profiles for the monophosphate of GMP compared with IMP in their respective NADP+complexes. These complexes undergo partial reactions that mimic different steps in the overall catalytic cycle. The relaxation profiles indicate that the substrate monophosphates have distinct interactions in E·IMP·NADP+and E·GMP·NADP+complexes. These findings were not anticipated by x-ray crystal structures, which show identical interactions for the monophosphates of GMP and IMP in several inert complexes. In addition, the motion of the cofactor is enhanced in the E·GMP·NADP+complex. Last, the motions of the substrate and cofactor are coordinately regulated; the cofactor has faster local motions than GMP in the deamination complex but is more constrained than IMP in that complex, leading to hydride transfer. These results show that field cycling can be used to investigate the dynamics of protein-bound ligands and provide new insights into how portions of the substrate remote from the site of chemical transformation promote catalysis.
From the Departments of Biology.
Masha M
MM
Rosenberg
Biochemistry, and.
Alfred G
AG
Redfield
the Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467-3860 mary.roberts@bc.edu.
Mary F
MF
Roberts
From the Departments of Biology, hedstrom@brandeis.edu.|Chemistry, Brandeis University, Waltham, Massachusetts 02453 and.
Lizbeth
0000-0001-8432-3322
L
Hedstrom
D055162:Biocatalysis; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050899:GMP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D007291:Inosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
D003067:Coenzymes; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D007291:Inosine Monophosphate; D009249:NADP; D050899:GMP Reductase
GMPR; biophysics; cofactor dynamics; dehydrogenase; enzyme catalysis; nuclear magnetic resonance (NMR); nucleoside/nucleotide biosynthesis