Luiza Loges

Date of Award

2021

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme present in all species that have thus far been tested (Vuillamy et al., 1988). G6PD is the rate limiting enzyme in the Pentose Phosphate Pathway (PPP) (Wang and Engel, 2009) and reduces NADP+ to NADPH through the oxidation of glucose-6-phosphate (G6P) to 6-phospho-gluconolactone (Hirono et al. 1989). The effectiveness of the G6PD enzyme is vital to the health and survival of an organism, as the PPP is one of the major NADPH production mechanisms, and NADPH protects cells from succumbing to oxidative stress (Wang and Engel, 2009). In the present study, characterization of the C. elegans orthologs of two known deficient human G6PD variants was performed. Production of the R252L and D60N C. elegans GSPD variants was done via E. coli plasmid expression. Purification of the mutant enzymes was performed on a nickel column, and enzyme kinetics parameters of the mutant GSPD enzymes were determined and compared to the wild type enzyme. From the Michaelis-Menten kinetics assay results, it was determined that the Vmax value for the R252L mutant enzyme was 5.9 * 10-3 ± 7.5 * 10-4 μmol/min, the KM value was 1050 ± 200 μM, and the specific activity was 1.2 ± 0.15 μmol/min/mg. For the D60N enzyme variant it was determined that the Vmax value was 3.2 * 10-3 ± 2.1 * 10 -4 μmol/min, the KM value was 80 ± 22 μM, and the specific activity was 6 ± 1.3 μmol/min/mg. Lastly, the Vmax value for the wild type enzyme was 2.2 * 10-3 ± 1.4 * 10 -4 μmol/min, the KM value was 100 ± 27 μM, and the specific activity was 11 ± 5.7 μmol/min/mg. These results diverge slightly from those previously published on the human orthologs of the two mutants. A 2013 study by Bendaoud et al. in which the human ortholog of the R252L GSPD mutant was first cited, as G6PD Tunisia, found that the mutant retained an estimated 25% of normal enzymatic activity while this study found that the R252L mutant had about 11% activity compared to the wild type. A 1988 study by Vuillamy et al. found that the Metaponto variant (the human ortholog of the D60N mutant) had a KM of 47 μM relative to G6P concentration which is a little over half that observed in this study. The same study found that the enzymatic activity of the mutant within red blood cells was between 14 and 39% of the wild type while the present study found the activity of the D60N mutant to be about 59% of the wild type. This novel data presents evidence of the deficiency of the C. elegans orthologs of the Tunisia and Metaponto variants of human G6PD, thus illustrating their promise as model systems for their human counterparts.

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