Date of Award

2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Roger Laine

Abstract

The goal of this research is to identify compounds which act as inhibitors for the synthesis of guanosine 5'-diphospho-L -fucose (GDP-fucose). The enzymes involved in this pathway are guanosine 5'-diphospho-D-mannose 4,6-dehydratase (GDP-D-mannose 4,6-dehydratase) and guanosine 5' -diphospho-4-keto-6-D-deoxymannose epimerase/guanosine 5'-diphospho-4-keto-6-L-galactose reductase (also known as FX protein and GDP-fucose synthetase). In this research project, GDP-mannose 4,6-dehydratse, the first enzyme in this pathway was studied. The plasmid DNA that contained the gene for GDP-mannose 4,6-dehydratase was transformed into E. coli bacteria; the enzyme was expressed and purified for use in this research. Before assaying the enzyme, a method of analysis for the products of the enzyme reaction had to be developed. This lead to the development of an HPLC method to analyze guanosine nucleotides and guanosine sugar nucleotides, which has not been previously described in the literature. Once the enzyme was purified and the assay developed, a study of the enzyme kinetics was initiated. The KM value for GDP-mannose 4,6-dehydratse calculated in this research project was 280 +/- 12.3 muM and the Vmax was determined to be 0.102 +/- 0.0008 mumoles/min. The potential inhibitor compounds were selected based on their structurally similarity to the intermediate of the enzyme reaction, GDP-4-keto-6-deoxymannose, as well as the end product, GDP-fucose. The compounds that have shown a significant inhibitory effect on GDP-mannose 4,6-dehydratase include GDP-fucose, 1-deoxyfuconojirimycin, GDP, fucose, 1-deoxymannojirimycin, and 1-deoxynojirimycin. Several compounds were screened that did not show a detectable effect on the GDP-mannose 4,6-dehydratase enzyme. These included UDP-galactose, UDP-glucose, ADP-ribose, and tunicamycin.

ISBN

9780493329321

Pages

86

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