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Date of Award

6-2003

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Professor Gerard D. Wright

Abstract

Aspartate kinase (AK) from Saccharomyces cerevisiae (AKsc) catalyzes the first step in the aspartate pathway responsible for biosynthesis of L-threonine, L-isoleucine, and L-methionine in fungi. Little was known about amino acids important for AKsc substrate binding and catalysis. Hypotheses about important amino acids were tested using site directed mutagenesis to substitute these amino acids with others having different properties. Steady state kinetic parameters and pH titrations of the variant enzymes showed AKsc-K18 and H292 to be important for binding and catalysis. Little was known about how the S. cerevisiae aspartate pathway kinases, AKsc and homoserine kinase (HSKsc), catalyze the transfer of the ϒ-phosphate from adenosine triphosphate (ATP) to L-aspartate or L-homoserine, respectively. Two transfer paths are possible as are a range of mechanisms, with two extremes. Both kinases were shown to directly transfer the ϒ-phosphate from ATP to the amino acid. Experimental evidence was consistent with an associative mechanism of phosphoryl transfer but did not rule out a dissociative mechanism. AKSc and HSKsc are good targets for inhibitors because they catalyze phosphoryl transfers between very similar substrates. An attempt was made to rationally design inhibitors to these kinases by linking the substrates with a variable length linker to create bisubstrate compounds. This strategy failed inhibit AKsc and HSKsc, however, one of the bisubstrate compounds was a good inhibitor of AKIII from Escherichia coli. Inhibition of any of the enzymes in the aspartate pathway would lead to reduced production of amino acids. A pathway assay was optimized to allow screening of chemical libraries in the hope of identifying inhibitors to the first four pathway enzymes. A high throughput screen of 1,000 compounds identified two compounds capable of inhibiting the assay, one of which was the best inhibitor identified for AKsc.

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Biochemistry Commons

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