Scanning the prime-Site substrate specificity of proteolytic enzymes: A novel assay based on ligand-Enhanced lanthanide ion fluorescence

• Published in: Bioorganic & medicinal chemistry letters Vol. 12; no. 24; pp. 3619 - 3623
• Main Authors: Barrios, Amy M., Craik, Charles S.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 16-12-2002; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Chymotrypsin - chemistry; Chymotrypsin - metabolism; Combinatorial Chemistry Techniques; Enzymes and enzyme inhibitors; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Hydrolases; Lanthanoid Series Elements - chemistry; Ligands; Methods; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; Peptide Library; Peptides - chemistry; Peptides - metabolism; Substrate Specificity; Fluorogenic substrate; Serine endopeptidases; Bovine; Peptides; Enzyme; Combinatorial chemistry; Peptidases; Chymotrypsin; Vertebrata; Mammalia; Position isomer; Enzymatic activity; Tetrapeptide; Proteolysis; Animal; Substrate specificity; Chemical compound library; Hydrolases; Peptide synthesis; Artiodactyla; Kinetics; Solid phase; Lanthanide Ions; Ungulata
• ISSN: 0960-894X; 1464-3405
• DOI: 10.1016/S0960-894X(02)00786-2

A novel method for assaying the substrate specificity of proteolytic enzymes has been developed utilizing ligand-enhanced lanthanide ion fluorescence. This approach was used to develop peptide libraries to probe substrate specificity in the prime sites of proteolytic enzymes. A positional scanning synthetic combinatorial library of fluorogenic peptides was synthesized and used to determine the extended prime site specificity of bovine α-chymotrypsin. The enzyme showed a preference for Lys and Arg in the P1′ position, rather broad specificity in the P2′ position, and a slight Arg specificity in the P3′ position. The specificity profile of bovine α-chymotrypsin agrees well with previously reported data, and the substrate library reported herein should provide valuable information about the prime site substrate specificities of other proteolytic enzymes as well. Furthermore, the continuous fluorogenic assay described may prove useful in analyzing the activity of other hydrolytic enzymes.
Graphic