PMID:2502187
| Citation |
Tokushige, M, Tsujimoto, N, Oda, T, Honda, T, Yumoto, N, Ito, S, Yamamoto, M, Kim, EH and Hiragi, Y (1989) Role of cysteine residues in tryptophanase for monovalent cation-induced activation. Biochimie 71:711-20 |
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| Abstract |
We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain. The results indicate that tryptophanase proteins of E. coli B/1t7-A and K-12 are identical. When cysteine residues in tryptophanase were chemically modified with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K+ and NH4+ was abolished. In consideration of our previous results that Cys-298 was selectively modified by SH reagents [Honda T. et al. (1986) J. Chromatogr. 371, 353-360], Cys-298 seems to have a close relation to the expression of the effect of monovalent cations. Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5'-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation. The results of the synchrotron small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 A irrespective of the presence or absence of the K+ ion. These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range. The above results are compatible with the possibility that Cys-298 is involved in the formation of "monovalent cation binding site" in the holoenzyme. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Cations, Monovalent/pharmacology; Cloning, Molecular; Cysteine; Enzyme Activation/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Genes; Genes, Bacterial; Lyases/metabolism; Scattering, Radiation; Spectrometry, Fluorescence; Sulfhydryl Compounds; Tryptophanase/genetics; Tryptophanase/metabolism |
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