EFFECTS OF MUTATIONS IN THE HINGE REGION OF SERPINS

An expression system for alpha1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha1-antitrypsin on its activity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation of Ala347 to threonine in alpha1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha1-antitrypsin. Cleavage within the reactive-site loop of wild-type alpha1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction. The hinge region of the reactive-site loop of serpins would appear to be important for the partitioning in favor of the stable complex.