Abstract
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.
Published Version
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