At the onset of the investigations described in this thesis progress was being made on the elucidation of the crystal structure of the <em>Azotobacter</em><em>vinelandii</em> lipoamide dehydrogenase. Also the gene encoding this enzyme was cloned in our laboratory. By this, a firm basis was laid to start site directed mutagenesis studies aimed at deepening the insight in the reaction mechanism of lipoamide dehydrogenase. At the start of this work no site directed mutated mutated lipoamide dehydrogenases were reported though for the <em>E. coli</em> enzyme some mutated enzymes were announced.<p>The first goal was to assess the function(s) of the active site base, histidine. Therefore the histidine was replaced by other residues and a glutamate, held responsible for affecting the pK <sub>a</sub> of the histidine was mutagenised. It was expected that<br/>the mutations would alter the reaction rates of the different reaction intermediates and that these intermediates could be identified and studied.<p>Chapter 2 describes the steady state spectral properties and in Chapter 3 the steady state and rapid reaction kinetics and some rapid reaction spectral properties of the wildtype and mutated enzymes are presented.<p>Since modeling studies suggested that Tyr16 was involved in substrate binding, this residue was replaced with other residues in order to stabilize the binding of the substrate, lipS <sub>2</sub> , and hence to be able to grow crystals of the enzyme with the substrate bound. The results obtained with these mutated enzymes are presented in Chapter 4.<p>Preliminary experiments with <em>A. vinelandii, E. coli</em> and <em>P. fluorescens</em> lipoamide dehydrogenase showed strong reactivity of the latter enzyme with antibodies raised against <em>A. vinelandii</em> enzyme while no cross reactivity was observed with the <em>E. coli</em> enzyme. This indicates that high sequence homology is present between the <em>A. vinelandii</em> and the <em>P. fluorescens</em> enzymes and that the <em>P.</em><em>fluorescens</em> enzyme constitutes a natural 'mutated' effective lipoamide dehydrogenase. Cloning and sequence analysis of this gene, described in Chapter 5 opens the way to site directed mutagenesis. In Chapter 6 a characterization of the <em>P. fluorescens</em> wild-type enzyme is presented.
|Qualification||Doctor of Philosophy|
|Award date||8 May 1992|
|Place of Publication||S.l.|
|Publication status||Published - 1992|
- molecular genetics