Random substitution of large parts of the propeptide of yeast proteinase A
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Random substitution of large parts of the propeptide of yeast proteinase A. / van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.
I: Journal of Biological Chemistry, Bind 270, Nr. 15, 1995, s. 8602-9.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Random substitution of large parts of the propeptide of yeast proteinase A
AU - van den Hazel, H B
AU - Kielland-Brandt, Morten
AU - Winther, Jakob R.
PY - 1995
Y1 - 1995
N2 - The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.
AB - The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.
KW - Amino Acid Sequence
KW - Aspartic Acid Endopeptidases
KW - Base Sequence
KW - Enzyme Precursors
KW - Molecular Sequence Data
KW - Mutation
KW - Oligodeoxyribonucleotides
KW - Peptide Fragments
KW - Plasmids
KW - Protein Precursors
KW - Protein Processing, Post-Translational
KW - Saccharomyces cerevisiae
KW - Saccharomyces cerevisiae Proteins
M3 - Journal article
C2 - 7721762
VL - 270
SP - 8602
EP - 8609
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -
ID: 43974368