Multifunctional Tryptophan-synthesizing Enzyme

Abstract

After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179 -190), we here describe structural and catalytic properties of the multifunctional tryptophansynthesizing enzyme. The kinetic parameters k cat of all five activities and K m for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, Nterminally blocked fragment with a molecular weight of 119,000. In addition, an increase in ␤-elimination accompanied by a decrease in ␤-replacement activity of the ␤-reaction during proteolysis was observed. In most procaryotes, tryptophan synthase is organized as a tetrameric bienzyme complex consisting of a central ␤ 2 -dimer (TRPS␤) combined with two peripheral ␣-subunits (TRPS␣). The Escherichia coli and Salmonella typhimurium enzymes in particular have been intensively investigated in the past (1, 2). In fungi such as Saccharomyces cerevisiae and Neurospora crassa, tryptophan synthase is a dimer of identical bifunctional polypeptides (3,4,5). In contrast, the enzyme isolated from Euglena gracilis, here designated as multifunctional tryptophan-synthesizing enzyme (MTSE), is capable of catalyzing all reactions shown in Scheme I. Although this discovery was made 2 decades ago (6, 7), no thorough further characterization of this multifunctional enzyme has been published so far, probably due to the excessive expenditure to cultivate sufficient amounts of E. gracilis and the difficulties of purifying MTSE with outdated technology. Using state of the art equipment and materials, we established a reliable and simple procedure to obtain MTSE from E. gracilis (8). Here we present both a reinvestigation of earlier initial findings and new data with respect to structural and functional properties of the enzyme. EXPERIMENTAL PROCEDURESChemicals, Enzymes, and E. coli Strains-If not otherwise stated, all chemicals (reagent or ultrapure grade) were obtained from Sigma (Deisenhofen, Germany) or Merck...