Role of cysteine residues in tryptophanase for monovalent cation-induced activation.
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.
Tokushige
M
M
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Tsujimoto
N
N
Oda
T
T
Honda
T
T
Yumoto
N
N
Ito
S
S
Yamamoto
M
M
Kim
E H
EH
Hiragi
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002414:Cations, Monovalent / Q000494:pharmacology*; D003001:Cloning, Molecular; D003545:Cysteine; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008190:Lyases / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D014368:Tryptophanase / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D013438:Sulfhydryl Compounds; D008190:Lyases; D014368:Tryptophanase; D003545:Cysteine