Interaction of human alpha 1-proteinase inhibitor with chymotrypsinogen A and crystallization of a proteolytically modified alpha 1-proteinase inhibitor.
Human alpha 1-proteinase inhibitor (alpha 1-PI) can form very stable complexes with chymotrypsinogen A or chymotrypsin if limited proteolysis by a contaminant proteinase is prevented with diisopropyl fluorophosphate. The contaminant proteinase cleaves the alpha 1-PI component in the alpha 1-PI-chymotrypsinogen A complex close to its N-terminus, between threonine-11 and aspartate-12 and the chymotrypsinogen A part between tyrosine-146 and threonine-147. By this modification the complex becomes unstable and dissociates into modified alpha 1-PI and neo-chymotrypsinogen A. A tritium labelling experiment shows that the contaminant proteinase is present in a 0.5-1.0% (w/w) ratio in the inhibitor preparation. These experiments indicate that alpha 1-PI is not a temporary inhibitor for these enzymes, as assumed by other authors. Isolated modified alpha 1-PI can be crystallized as tetragonal bipyramides from 2.6M sodium potassium phosphate pH 8.0. The crystals are suitable for three dimensional X-ray structure analysis. In spite of the cleavage of the susceptible peptide bond by chymotrypsinogen A, the C-terminal 3.6 kDa cleavage peptide remains tightly bound to the inhibitor by means of non-covalent interactions. In accordance with the result of the known complete amino-acid sequence of the inhibitor this finding offers an alternative explanation to the suggestion of alpha 1-PI being a double headed inhibitor. Isolated neo-chymotrypsinogen A can be activated to active chymotrypsin and can form a very labile 1 : 1 complex with alpha 1-PI, which dissociates rapidly into inactive inhibitor and neo-chymotrypsinogen.
D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002919:Chymotrypsinogen / Q000378:metabolism*; D003460:Crystallization; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D014961:X-Ray Diffraction; D000515:alpha 1-Antitrypsin
D001798:Blood Proteins; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; D002919:Chymotrypsinogen