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