Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/67694
標題: Effects of C-Terminal Truncation on Autocatalytic Processing of Bacillus licheniformis gamma-Glutamyl Transpeptidase
作者: Chang, H.P.
Liang, W.C.
Lyu, R.C.
Chi, M.C.
Wang, T.F.
Su, K.L.
Hung, H.C.
Lin, L.L.
關鍵字: Bacillus licheniformis
gamma-glutamyl transpeptidase
C-terminal
truncation
autocatalytic processing
analytical ultracentrifugation
escherichia-coli
helicobacter-pylori
autoproteolytic activation
glutathione metabolism
crystal-structures
protein
intermediate
resistance
threonine
mechanism
期刊/報告no:: Biochemistry-Moscow, Volume 75, Issue 7, Page(s) 919-929.
摘要: The role of the C-terminal region of Bacillus licheniformis gamma-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581-585, 577-585, 576-585, 566-585, 558-585, 523-585, and 479-585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-gamma-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Delta (576-585), pro-Delta (566-585), and pro-Delta(558-585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Delta(577-585), Delta(576-585), Delta(566-585), Delta(558-585), Delta(523-585), and Delta(479-585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and. Delta G(0) for the unfolding. BlGGT-4aa and BlGGT sedimented at similar to 4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f(o)) of BlGGT-4aa, BlGGT, Delta(581-585), and Delta(577-585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.
URI: http://hdl.handle.net/11455/67694
ISSN: 0006-2979
文章連結: http://dx.doi.org/10.1134/s0006297910070151
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