Selection of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a phage display peptide library.
A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against alpha-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with alpha-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of alpha-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for alpha-chymotrypsin was estimated to be 10(-6) M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified alpha-chymotrypsin than for natural alpha-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to alpha-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe- Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of alpha-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.
Department of Pure and Applied Biochemistry, Lund University, Sweden.
M
M
Krook
C
C
Lindbladh
J A
JA
Eriksen
K
K
Mosbach
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013552:Swine
D009842:Oligopeptides; D019151:Peptide Library; D010456:Peptides, Cyclic; D015842:Serine Proteinase Inhibitors; D002918:Chymotrypsin