Kristallstrukturuntersuchungen zum Katalyse- und Regulationsmechanismus der Tyrosin-regulierten 3-Deoxy-D-arabino-Heptulosonat-7-Phosphat-Synthase aus Saccharomyces cerevisiae

Abstract

The ability to synthesize the aromatic amino acids tyrosine, phenylalanine and tryptophan is confined to prokaryotes, yeast, algae and plants. The first common step in biosynthesis for these amino acids follows the shikimate pathway and the initial reaction, the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) is catalyzed by the enzyme family of 3-Deoxy-arabino-heptulosonate-7-phosphate synthases (DAHP synthases). These isoenzymes require a divalent metal ion and most of them are feedback regulated by the respective end product.This thesis describes studies on crystal structures of the tyrosine regulated DAHP synthase from Saccharomyces cerevisiae and a variety of its complexes. The binding pockets for both substrates could be determined on the basis of the double complex of the DAHP synthase with manganese, PEP and the E4P analogue glyceraldehyde-3-phosphate (G3P). Comparisons of complexes with different metal ions, with and without PEP, with the PEP analogue P2G or the E4P analogue G3P allow to draw conclusions about the role of the metal ion and the substrate PEP for catalysis and about the stereo specific reaction mechanism. A complex of DAHP synthase, manganese(II) and tyrosine reveals the effector binding pocket of this enzyme family. By comparing the active and the inhibited structures of the enzyme the inhibition mechanism of the tyrosine sensitive DAHP synthase could be understood. The structure of the point mutant Gly226Ser was also determined and comparison with the tyrosine complex of the wild type reveals the change of its feedback inhibition from tyrosine to phenylalanine sensitivity caused by this single amino acid exchange.