Untersuchungen zu Struktur und Funktion von Transketolase und Transaldolase, sowie biochemische Charakterisierung der Enzyme aus Escherichia coli

Abstract

Transketolase aqnd Transaldolase are enzymes of the non-oxidative pentosephosphate pathway and catalyze the reversible transfer of C$_{2}$- and C3-fragments between different sugarphosphates. Transketolase was purified by fractionated ammonia sulfate precipitations and anion exchange columns to homogeneity with a yield of45%. The specific activity oftransketolase was 110 U/mg. Besides the physiological sugarphosphates also unphosphorylated sugar derivates as deoxy-, nitro and acidosugars were accepted with rates up to 18 U/mg.Crystals from apo- and holotransketolase were obtained and X-ray datasets were measured to a resolution of 2.0 angström. With the knowledge of the transketolase structure from yeast investigations in the $\textit{E.coli}$ transketolase substrate channel were performed. Arg359, Ser386 and Arg521 are involved in binding the phosphate group of the substrate. Asp470 is neccessary for binding the C2-hydroxyl group of the acceptor sugars. Site specific mutations at position Asp470 lead to mutants that accepted pyridinecarbaldehyds. With a His103 yeast transketolase mutant for the first time pyruvate was a donor substrate for transketolase. Transaldolase was purified purified by fractionated ammonia sulfate precipitations and anion exchange columns tohomogeneity with a yield of 50%. High conversion rates were obtained for transaldolase only with the physiological substrates (80 U/mg), with unphosphorylated substrates rates of 8 U/mg were the maximum. With KBH4-reduction a stable enzyme-substrate-complex oftransaldolase could be synthesized. The 3D-structure of native transaldolase and the complex was solved in cooperation with the group of Prof. Schneider, Stockholm. Transaldolase has as the related aldolases an alpha/beta barrel structure. From the X-ray structure site directed mutagenesis of the actice centre (Asp17, Asn35, Ser176) and the dimerisation area (Arg300) were performed. Exchanges of Asp17 and Asn35 resulted in an almost complete loss ofactivity. A change ofresidue Ser176 lead to a five fold decrease in the affinity towards the donor substrate. An exchange of Arg300 lead to a monomeric enzyme at pH 8.5, however, the activity and the stability of the transaldolase are unaffected