Expression of rat chymotrypsinogen in yeast: a study on the structural and functional significance of the chymotrypsinogen propeptide

The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not im...

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Bibliographic Details
Published in:FEBS letters 1996-01, Vol.379 (2), p.139-142
Main Authors: Venekei, István, Gráf, László, Rutter, William J.
Format: Article
Language:English
Subjects:
4-methylumbelliferyl p-(N N,N-trimethyl ammonium) cinnamate chloride
4-methylumbelliferyl p-guanidinobenzoate
7-amino-4-methylcoumarin
AMC
Amino Acid Sequence
Animals
Base Sequence
Cattle
Cell Line
Chymotrypsin
Chymotrypsinogen - biosynthesis
Chymotrypsinogen - chemistry
Chymotrypsinogen - metabolism
Cloning, Molecular
Disulfide bond
Disulfides
DNA Primers
Dogfish
Dogs
Enzyme Activation
Gene Library
Goats
Horses
Kinetics
Molecular Sequence Data
MUGB
Mutagenesis, Site-Directed
MUTMAC
p-nitroanilide
pNA
Polymerase Chain Reaction
Protein folding
Rats
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Saccharomyces cerevisiae
SBTI
Sequence Homology, Amino Acid
Sheep
soybean trypsin inhibitor
Substrate Specificity
tosyl-l-alanyl chloromethyl ketone
TPCK
Trypsinogen - chemistry
Zymogen activation
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Summary:The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non-specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non-specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen.
ISSN:0014-5793
1873-3468
DOI:10.1016/0014-5793(95)01483-7