Kinetic Characterization of Caenorhabditis elegans Glucose-6-Phosphate Dehydrogenase

Angel Jordan

Date of Award

2017

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human metabolic mutation, affecting more than 400 million people worldwide. G6PD catalyzing the first step in the pentose phosphate pathway oxidizes β-D-glucose 6-phosphate (G6P) to 6-phosphoglucono-δ-lactone with the simultaneous reduction of NADP+ to NADPH. Lack of production of NADPH results in oxidative stress in human blood cells, leading to hemolysis. Disruption of G6PD catalysis can also result in embryogenic effects in animals due to a lack of nucleotides. This study used the nematode Caenorhabditis elegans as a model organism to characterize the G6PD worm enzyme. C. elegans has gained popularity as a research model. No previous studies characterizing G6PD kinetics in C. elegans have been reported. C. elegans G6PD inserted into the vector pET303/CT-His was partially purified from E. coli Rosetta cells by nickel affinity chromatography. pH, temperature, cation preference, and substrate kinetics (NADP+ and G6P) were investigated to optimize kinetic assay conditions. An optimal pH of 9 and an optimal temperature of 45 °C were found. Preference for Mg2+ as a cation was detected but more data is needed to confirm this. A KM of 72 ± 12 μM for NADP+, and a KM of 80 ± 8 μM for G6P were calculated from the Michaelis Menten equation fit to the enzyme kinetics data. Specific activity was calculated to be within the range 5-18 U/mg.

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