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Volume 272, Number 16, Issue of April 18, 1997 pp. 10616-10623
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Multifunctional Tryptophan-synthesizing Enzyme
THE MOLECULAR WEIGHT OF THE EUGLENA GRACILIS PROTEIN IS UNEXPECTEDLY LOW

(Received for publication, December 10, 1996, and in revised form, February 6, 1997)

Thomas Schwarz , Katharina Uthoff ^¶ , Claudia Klinger ^¶ , Helmut E. Meyer , Peter Bartholmes ^¶ and Michael Kaufmann ^¶

From the ^¶ Institut für Biochemie, Universität Witten/Herdecke, Stockumer Str. 10, 58453 Witten, Germany, the  Institut für Physiologische Chemie I, Ruhruniversität Bochum, Universitätsstr. 150, 44780 Bochum, Germany, and  bitop GmbH Witten, Stockumer Str. 10, 58453 Witten, Germany

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 tryptophan-synthesizing 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, N-terminally 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.

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