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| Home > Publications database > Untersuchungen zur Regulation des Pentosephosphatweges in Corynebacterium glutamicum |
| Home > Publications database > Untersuchungen zur Regulation des Pentosephosphatweges in Corynebacterium glutamicum |
| Dissertation / PhD Thesis/Book | PreJuSER-26988 |
2000
Forschungszentrum, Zentralbibliothek
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/20587
Report No.: Juel-3743
Abstract: Since many years the gram-positive bacterium Corynehacterium glutamicum is used for the technical production of amino acids like L-glutamate and L-lysine . As for the Biosynthesis of these amino-acids also NADPH is needed, the purpose of this doctorial dissertation was to study the regulation of the pentose phosphate pathway. The firnt two enzymes of the pentose phosphate pathway, the glucose-6-phosphate dehydrogenase and the 6-phosphogluconate dehydrogenase were isolated from C. glutamicum and characterized . According to biochemical investigations, the glueose-6-phosphate dehydrogenase consists of two different protein-subunits, which are encoded by the adjacent zwf- and opcA-genes. The glucose-6-phosphat-dehydrogenase operates according to an ordered Bi-Bi-mechanism, whereas the 6-phosphogluconate dehydrogenase operates according to a Theorell-Chance ordered Bi Ter mechanism . the case of both enzymes NADPH competes with NADP for the cofactor binding site, whereby the competitive inhibition constants of NADPH are similar to the Michaelis-constants of NADP ; these constants are all around 15 pM to 40 pM . As the intracellular concentrations of NADP and NADPH are between 100 pM and 370 pM and therefore significantly greater than the corresponding kinetic constants, both enzymes are influenced by the NADPH/NADP-concentration ratio in vivo. In the case of a leucineauxotrophic strain of C. glutamicum, this ratio increased fourfold, which showed that the NADPH/NADP-concentration ratio is very important for the in vivo regulation of the pentose phosphate pathway. On the Basis of the kinetic dato a mode] was deduced, which allows the calculation of the in vivo activity of the glucose-6-phosphate dehydrogenase from the intracellular concentrations of glucose-6-phosphate, NADP and NADPH . The obtained specific activities of around 10xI0i3 units per mg dry weight were in full accord with the same fluxes determined by NMR after "C-Iabelling. The in vivo activity of the glucose-6-phosphate-dehydrogenase was a factor of 4 to 8 smaller than the maximal in vitro activity in the case of substrate saturation . As a smaller intracellular NADPH/NADP-concentration-ratio leads to an increased activity of both dehydrogenases of the pentose phosphate pathway, it is likely, that NADPH does not limit lysine production of C .glutamicum
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