A study of the specificity of barley chymotrypsin inhibitor 2 by cysteine engineering of the P1 residue.
A combination of oligonucleotide-directed mutagenesis and chemical modification was used to produce reactive site (P1) variants of chymotrypsin inhibitor II (CI2) in an attempt to create more potent inhibitors and examine inhibitory specificity. Mutagenesis to introduce a unique cysteine (CI2M59C) followed by modification to S-carboxamidocysteine with iodoacetamide produced a 7-fold more potent inhibitor of subtilisin BPN' than the wild type inhibitor. Modification with iodoacetic acid, which gives a negatively charged P1 residue (S-carboxymethylcysteine), generates a weaker inhibitor of subtilisin BPN' and chymotrypsin. Further chemical modification experiments of CI2M59C with a series of iodoalkanes of increasing chain lengths was used to determine the optimal P1 side chain length required for potent inhibition of porcine pancreatic elastase. A trend was observed which implies that for CI2 the original methionine residue or its isostere S-ethylcysteine are most effective residues at this position and not alanine as might have been expected from the substrate specificity. The mutant CI2M59C did not inhibit human neutrophil elastase. The iodoalkane modifications not only resulted in recovery of inhibitory activity but also proved to be substantially more potent inhibitors of human neutrophil elastase than wild-type CI2.
Hasan
Z
Z
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, UK.
Leatherbarrow
R J
RJ
D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D003545:Cysteine / Q000737:chemistry; D001467:Hordeum / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity
D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D002918:Chymotrypsin; D003545:Cysteine