Activation of Bean (Phaseolus vulgaris) α-Amylase Inhibitor Requires Proteolytic Processing of the Proprotein

8 pages, figures, and tables statistics.
Seeds of the common bean (Pbaseolus vulgaris) contain a plant defense protein that inhibits the a-amylases of mammals and insects. This a-amylase inhibitor (aAl) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M,) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Pbaseolus, we found that antibodies to uAl recognize large (M. 30,000-35,000) polypeptides as well as typical aAl processing products (M, 15,000- 18,000). aAl activity was found in all extracts that had the typical aAl processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant aAl in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-aAl when the gene is expressed in tobacco. When pro-aAl was separated from mature aAl by gel filtration, pro-aAl was found not to have a-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.