A kinetic mechanism for the polymerization of alpha(1)-antitrypsin

The mutation in the Z deficiency variant of alpha(1)-antitrypsin perturbs t he structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepat ocytes to form inclusions that are associated with neonatal hepatitis, juve nile cirrhosis, and hepatocellular carcinoma. The process of polymer format ion has been investigated here by intrinsic: tryptophan fluorescence, fluor escence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoac etamide. These biophysical techniques have demonstrated that alpha(1)-antit rypsin polymerization is a two-stage process and have allowed the calculati on of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the s low phase is bimolecular and associated with loop-sheet interaction and pol ymer formation. The naturally occurring Z, S, and I variants and recombinan t site-directed reactive loop and shutter domain mutants of alpha(1)-antitr ypsin were used to demonstrate the close association between protein stabil ity and rate of alpha(1)-antitrypsin polymerization, Taken together, these data allow us to propose a kinetic mechanism for alpha(1)-antitrypsin polym er formation that involves the generation of an unstable intermediate, whic h can form polymers or generate latent protein.