Kinetic Characterization of Caenorhabditis elegans postembryonic Glyceraldehyde-3-Phosphate Dehydrogenase

Ruth Silimon

Date of Award

2013

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Kinetics, C. Elegans, Glyceraldehyde-3-Phosphate Dehydrogenase

Area of Concentration

Chemistry, Biology

Abstract

Caenorhabditis elegans is a model organism in modern biological research. The worm has four different genes, gpd 1-4, for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH catalyzes the oxidation and phosphorylation of glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) using NAD+ and Pi as substrates. The 1,3-BPG product is used in a later reaction in glycolysis to generate ATP. The physiological significance of multiple GAPDH isoenzymes within C. elegans is still not understood. This is the first project to characterize partially purified GPD-3 formally. The IMPACT purification system from NEB was used in conjunction with Rosetta (DE3) cell hosts to over-express and partially purify the enzyme. Enzyme kinetics were performed with all three substrates at pH 8.0. The Km values for NAD+, G3P, and Pi were estimated to be 0.3 mM, 2.6 mM, and 0.4 mM, respectively. The enzyme's specific activity was found to be within the range of 3.6 to 5.3 µmol/min•mg. The Km for NAD+ reported here is lower than the value found for an endogenous GPD mixture however, the Km for G3P is much higher, suggesting the need for further analysis.

Rights

The author has not granted New College of Florida the nonexclusive right to archive, make accessible, and distribute for educational purposes this work in whole or in part in all forms of media, now or hereafter known. The copyright of this work remains with the author.

This document is currently not available here.

Share

COinS