The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling

• Published in: Sheng wu hua hsüeh yü sheng wu wu li hsüeh pao Vol. 33; no. 6; p. 591
• Main Authors: Fei, H, Luo, M J, Ye, Y Z, Ding, D F, Chi, C W
• Format: Journal Article
• Language: English
• Published: China 2001
• ISSN: 0582-9879

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L). Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P(3) site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.