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

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

Disruption of Escherichia coli transaldolase into catalytically active monomers : evidence against half-of-the-sites mechanism

AbstractDisruption of the hydrogen bonding network at the interface of Escherichia coli transaldolase by substitution of R300 to a glutamic acid residue resulted in a monomeric enzyme at basic pH values, with almost no change in the kinetic parameters. The stability of the R300A and R300E mutants towards urea and thermal inactivation is similar to that of the wild-type enzyme. X-ray analysis showed that no structural changes occurred as a consequence of the side chain replacement. This indicates that the quaternary structure is not required for catalytic activity nor does it contribute significantly to the stability of the enzyme. The results are not consistent with a proposed half-of-the-sites reaction mechanism

Modeling of reaction kinetics for reactor selection in the case of L-erythrulose synthesis

To choose the most effective process design in enzyme process development it is important to find the most effective reactor mode of operation. This goal is achieved by modeling of the reaction kinetics as a tool of enzyme reaction engineering. With the example of the transketolase catalyzed L-erythrulose synthesis we demonstrate how the most effective reactor mode can be determined by kinetic simulations. This is of major importance if the biocatalyst deactivation is caused by one of the substrates as in this case by glycolaldehyde. The cascade of two membrane reactors in series with soluble enzyme is proposed as a solution for the enzyme deactivation by one of the substrates

Identification of catalytically important residues in the active site of Escherichia coli transaldolase

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A. E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations. N35A, E96A, and T156A resulted mainly in an effect on apparent k(cat), with little changes in apparent K-m values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent K-m value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent K-m value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in k(cat), and a fivefold increase in the apparent K-m value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate