Expression and kinetic characterization of barley chymotrypsin inhibitors 1a and 1b.
The genes for chymotrypsin inhibitors 1a and 1b (CI-1a and CI-1b) from barley have been expressed in E. coli, and the CI-1a and CI-1b proteins purified. These proteins, although highly homologous, differ in the active site region at P2, P1' and P3' (Schechter and Berger nomenclature), and so might be expected to have differing specificities. Despite this, analysis of the inhibition kinetics showed that each displayed very similar kinetic behaviour when tested against a range of proteinases. The specificity of the CI-1 proteins is different to that of the other main barley inhibitor, CI-2, and Ki values are found to follow the series subtilisin Carlsberg < neutrophil elastase approximately subtilisin BPN' << chymotrypsin. Only very weak inhibition is found of trypsin, and pancreatic elastase is not measurably inhibited. For the proteinases inhibited most strongly, characteristic slow-binding inhibition kinetics were observed, whereas classical inhibition applied to the weaker interactions. The results are consistent with the major determinant of specificity being the P1 residue of the inhibitor, which is the same in both CI-1a and CI-1b. Consistent with this, is the similar spectrum of specificity found for the homologous inhibitor eglin c from leech, which has the same P1 residue. Both the CI-1 proteins are found to be less stable than CI-2, with CI-1a being significantly less stable than CI-1b as measured by guanidinium hydrochloride unfolding experiments. Possible reasons for the reduced stability are discussed in view of the sequence differences between CI-1a, CI-1b and CI-2.
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, UK.
M A
MA
Greagg
A B
AB
Brauer
R J
RJ
Leatherbarrow
D000595:Amino Acid Sequence; D001483:Base Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; C088416:chymotrypsin inhibitor protein, Hordeum vulgare; D002918:Chymotrypsin