The Inhibitory
Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using
Site-directed Mutagenesis and Molecular Modeling
FEI Hao, LUO Ming-Juan, YE Yu-Zheng1, DING Da-Fu1,
CHI Cheng-Wu*
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry and Cell Biology, the Chinese Academy of Sciences, Shanghai 200031,
China；1Shanghai Institute of
Biochemistry and Cell Biology, the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract 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 P1,
P2 and P4 of eglin C with a basic residue using protein
engineering could make eglin C a very strong inhibitor for furin (Ki
around 10-9 mol/L),and even more strong for kexin (Ki
around 10-11 mol/ L). Results indicated that： (1) A basic residue Lys or
Arg at P1 site is prerequisite for the inhibitor. (2) The second
mutation with basic residue at P4 site drastically increase the
inhibitory activity by two orders of magnitude. (3) A basic residue at P2
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 P3 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.
Key words proprotein convertase； proteinase inhibitor； site-directed mutagenesis； molecular modeling
*Corresponding author： Tel, 86-21-64337152； Fax, 86-21-64338357； e-mail, [email protected]