Multifunctional tryptophan-synthesizing enzyme. The molecular weight of the Euglena gracilis protein is unexpectedly low.
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 beta-elimination accompanied by a decrease in beta-replacement activity of the beta-reaction during proteolysis was observed.
Schwarz
T
T
Institut für Biochemie, Universität Witten, Herdecke, Stockumer Str. 10, 58453 Witten, Germany.
Uthoff
K
K
Klinger
C
C
Meyer
H E
HE
Bartholmes
P
P
Kaufmann
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D005056:Euglena gracilis / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D014357:Trypsin; D014367:Tryptophan Synthase