Faculty Mentor
Dr. Nicholas E. Burgis
Presentation Type
Poster
Primary Discipline of Presentation
Chemistry and Biochemistry
Abstract
Two of the major nucleotide bases in DNA and RNA (Adenine and Guanine) are derived from the purine Inosine Monophosphate (IMP). IMP may occasionally form the noncanonical nucleotide Inosine Triphosphate (ITP) within the cell and become incorporated into DNA during replication, leading to potentially lethal errors. To combat this, human cells produce the “housekeeping” enzyme Inosine Triphosphatase (ITPA) to revert ITP to IMP. A mutation of this protein that replaces the 178th amino acid Arginine with Cysteine (R178C) is associated with a fatal infantile encephalopathy. Previous assessments of enzyme-substrate binding and catalysis for ITPA variants have been run at a neutral pH of 7.4, but a preliminary survey performed last year has implied that a pH of 6.5 may increase the enzyme catalysis of the R178C variant when tested via High-Pressure Liquid Chromatography (HPLC) (Nieffenegger et al., 2025). To produce protein for these assays, we lengthened the time that the cells we used spend with the inducer molecule IPTG to overnight (18-24 hours) instead of the established 4-hour exposure. We have compared enzyme performance at these pHs via Michaelis Menten kinetics. Similar assays were also be used to compare the enzymatic activity of our proteins when flash-frozen in liquid nitrogen to our established method of storage in glycerol. Changes in activity resulting from alterations to assay pH or protein storage conditions will be useful for the development of a high throughput assay to identify potential stabilizing agents.
Recommended Citation
Reisnouer, Jared J. and Pines, Wally D., "Optimizing ITPA R178C Assays via HPLC" (2026). 2026 Symposium. 1.
https://dc.ewu.edu/srcw_2026/works_2026/works_2026/1
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
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Optimizing ITPA R178C Assays via HPLC
Two of the major nucleotide bases in DNA and RNA (Adenine and Guanine) are derived from the purine Inosine Monophosphate (IMP). IMP may occasionally form the noncanonical nucleotide Inosine Triphosphate (ITP) within the cell and become incorporated into DNA during replication, leading to potentially lethal errors. To combat this, human cells produce the “housekeeping” enzyme Inosine Triphosphatase (ITPA) to revert ITP to IMP. A mutation of this protein that replaces the 178th amino acid Arginine with Cysteine (R178C) is associated with a fatal infantile encephalopathy. Previous assessments of enzyme-substrate binding and catalysis for ITPA variants have been run at a neutral pH of 7.4, but a preliminary survey performed last year has implied that a pH of 6.5 may increase the enzyme catalysis of the R178C variant when tested via High-Pressure Liquid Chromatography (HPLC) (Nieffenegger et al., 2025). To produce protein for these assays, we lengthened the time that the cells we used spend with the inducer molecule IPTG to overnight (18-24 hours) instead of the established 4-hour exposure. We have compared enzyme performance at these pHs via Michaelis Menten kinetics. Similar assays were also be used to compare the enzymatic activity of our proteins when flash-frozen in liquid nitrogen to our established method of storage in glycerol. Changes in activity resulting from alterations to assay pH or protein storage conditions will be useful for the development of a high throughput assay to identify potential stabilizing agents.
