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Identification of The Binding Site for Ammonia in GMP Reductase
Author(s) -
YAO Tianjiong,
Rosenberg Masha,
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.722.10
Subject(s) - chemistry , ammonia , deamination , guanosine monophosphate , hydride , stereochemistry , active site , guanosine , binding site , cofactor , biochemistry , nucleotide , enzyme , organic chemistry , hydrogen , gene
The reaction of guanosine monophosphate reductase (GMPR) includes two steps: (1) a deamination step that releases ammonia from GMP and forms the intermediate E‐XMP*; (2) a hydride transfer step that converts E‐XMP* to IMP utilizing NADPH as a cofactor. The hydride transfer step is the rate limiting step, yet we failed to observe a burst of ammonia release. This observation suggests that ammonia remains bound to the enzyme during the hydride transfer step. We identified a possible ammonia binding site by inspection of crystal structure of human GMPR type 2. Three candidate amino acids were selected and probed by site directed mutagenesis. The substitution of all 3 residues decreased intermediate production about 4 fold but decreased the reaction with ammonia by at least 100 fold. Therefore, these substitutions behave as expected for mutations at the ammonia binding site.