Fluorescent Peptidyl Substrates as an Aid in Studying the Substrate Specificity of Human Prohormone Convertase PC1 and Human Furin and Designing a Potent Irreversible Inhibitor
| Autor: | Claude Lazure, Ajoy Basak, Nabil G. Seidah, Alain Boudreault, François Jean |
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| Rok vydání: | 1995 |
| Předmět: |
endocrine system
Stereochemistry Molecular Sequence Data Prohormone convertase Cleavage (embryo) Biochemistry Catalysis Furin activity Substrate Specificity Coumarins Aspartic Acid Endopeptidases Humans Amino Acid Sequence Subtilisins Enzyme kinetics Molecular Biology Furin Fluorescent Dyes biology Chemistry Hydrolysis Substrate (chemistry) Cell Biology Fluorescence enzymes and coenzymes (carbohydrates) biology.protein Substrate specificity Proprotein Convertases Oligopeptides |
| Zdroj: | Journal of Biological Chemistry. 270:19225-19231 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.270.33.19225 |
| Popis: | The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin. |
| Databáze: | OpenAIRE |
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