Identification of The Binding Site for Ammonia in GMP Reductase

Abstract

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.