The role of the hinge region of the beta 2-subunit in beta-replacement specificity of tryptophan synthase from Escherichia coli. Analysis of proteolytically modified beta species cleaved by endoproteinase Glu-C.
The beta 2-subunit of tryptophan synthase from Escherichia coli was proteolyzed between Glu-296 and Ser-297 (Glu-nicked beta 2), using staphylococcal protease V-8. Recombining Glu-nicked beta 2 with native alpha-subunits yields an enzymatically active and stable complex (alpha 2.Glu-nicked beta 2) with Kd = 3 microM. Comparing the properties of alpha 2.Glu-nicked beta 2 with those of isolated native beta 2-subunits revealed that the corresponding beta-chains are almost identical with respect to enzymatic specificities as well as to some spectral characteristics. The ratio of beta-replacement to beta-elimination activity is approximately 0.6 for both species. In the presence of L-serine, alpha 2.Glu-nicked beta 2 as well as native beta 2 exhibits "aqua" fluorescence emission at 500 nm, and no "amber" absorption at 468 nm can be detected with both species. However, when ammonium ions are added, the alpha 2.Glu-nicked beta 2 complex dramatically alters its properties. Similar to the native alpha 2 beta 2 enzyme, the ratio of beta-replacement/beta-elimination activity increases by a factor of about 10, the aqua fluorescence disappears, and the amber complex becomes detectable. We conclude that the continuity of the hinge region in the tryptophan synthase beta 2-subunit plays a crucial role in the alpha-mediated structural alteration creating beta-replacement specificity. The addition of NH4+ ions to the alpha 2.Glu-nicked beta 2 complex partially restores the structure-function relationships as described for native alpha 2 beta 2 tryptophan synthase.
Linkens
H J
HJ
Institut für Biochemie, Universität Witten/Herdecke, Federal Republic of Germany.
Bartholmes
P
P
Kaufmann
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*
D005971:Glutamates; D046911:Macromolecular Substances; D018698:Glutamic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014367:Tryptophan Synthase; D012695:L-Serine Dehydratase