Identification of the binding site for ammonia in GMP reductase

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dc.contributor.advisor Hedstrom, Lizbeth Yao, Tianjiong 2015-03-02T16:04:39Z 2015-03-02T16:04:39Z 2015
dc.description.abstract The overall reaction of guanosine monophosphate reductase (GMPR) converts GMP to IMP by using NADPH as a cofactor and it includes two sub-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 along with the oxidation of NADPH. The hydride transfer step is the rate limiting step, yet we failed to observe a burst of ammonia release. Meanwhile ammonia cannot stay in the same place where it is formed otherwise it will block NADPH. This observation suggests that ammonia remains bound to the enzyme during the hydride transfer step and there exists ammonia holding site after its release from the formation site. We identified a possible ammonia holding site by inspection of crystal structure of human GMPR type 2. Three candidate amino acids were selected and probed by site directed mutagenesis. The substitutions of all three residues decreased the reduction of GMP at least 50 fold and the oxidation of IMP at least 40 fold, and reduced the intermediate production at least 2 fold. Therefore, these substitutions behave as expected for mutations at the ammonia holding site.
dc.description.sponsorship Brandeis University, Graduate School of Arts and Sciences
dc.format.mimetype application/pdf
dc.language English
dc.language.iso eng
dc.publisher Brandeis University
dc.relation.ispartofseries Brandeis University Theses and Dissertations
dc.rights Copyright by Tianjiong Yao 2015
dc.subject GMP reductase
dc.subject ammonia binding site
dc.subject steady state
dc.subject pre-steady state
dc.title Identification of the binding site for ammonia in GMP reductase
dc.type Thesis
dc.contributor.department Graduate Program in Molecular and Cell Biology MS Masters Molecular and Cell Biology Brandeis University, Graduate School of Arts and Sciences

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