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 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.
Venekei
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Hormone Research Institute, University of California San Francisco 94143-0534, USA.
Gráf
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Rutter
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002919:Chymotrypsinogen / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides; D004284:Dogfish; D004285:Dogs; D004789:Enzyme Activation; D015723:Gene Library; D006041:Goats; D006736:Horses; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013379:Substrate Specificity; D014362:Trypsinogen / Q000737:chemistry
D017931:DNA Primers; D004220:Disulfides; D011994:Recombinant Proteins; D014362:Trypsinogen; D002919:Chymotrypsinogen