Dissecting the role of disulfide bonds for folding and enzyme-inhibitory activity of the Bowman-Birk inhibitor.


Peter Flecker


Institute of Physiological Chemistry and Pathobiochemistry, Gutenberg University Mainz, Germany


The Bowman-Birk inhibitor (BBI) from the soybean is a bifunctional inhibitor of trypsin and chymotrypsin/elastase characterized by a highly conserved array of 7 disulfide bonds within a relatively short polypeptide-chain of only 71 amino acids. The unusually high content of 20% of cysteines in the polypeptide chain of BBI suggests that this protein may also act as a biological storage of sulfur. The structure of BBI is characterized by a binary arrangement of an amino-terminal trypsin-reactive subdomain and a respective chymotrypsin-reactive subdomain. We have used the double-headed arrangement in the structure of BBI for assessing the functional and conformational integrity of the variants by comparative titration and activity determination experiments with trypsin. The trypsin-reactive subdomain of BBI is stabilized by 4 and the chymotrypsin-reactive subdomain by 3 S-S bonds. Interdomain disulfide linkages are not present in this molecule.

The structure of BBI displays structural peculiarities as exposed hydrophobic patches and buried ion pairs solvated by internal water molecules in the interdomain boundary. These features are in marked contrast to most other proteins comprising a hydrophobic core and exposed polar amino acids. The exposed hydrophobic patches are a structural peculiarity that is reminiscent of the kind of structural features that have been postulated to occur in partially folded proteins. We have hypothesized, that a hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core could be prevented by the array of seven disulfide bridges.

We have assessed the consequences of mutations in the conserved framework of S-S bonds in the trypsin-reactive subdomain for the autonomous refolding competence and the enzyme-inhibitory activity of the other subdomain directed against chymotrypsin. The deletion of disulfide bonds induces dramatic effects on the refolding competence not only of the trypsin-reactive subdomain, but also on the activity of the chymotrypsin-reactive subdomain. The variants may be distinguished into two categories. The first one displays only local irregularities as expected by hierarchical models of protein folding. In the mutants belong to the second category the chymotrypsin-inhibitory subdomain is also affected significantly in its refolding competence and its activity as a result of the mutations in the trypsin-inhibitory subdomain. Since the mutants belonging to the second category are located near ionic residues interacting across the interdomain boundary with ionic residues with opposite charge we have concluded that ionic interactions may be crucial for the kinetically controlled process of protein folding in addition to the hydrophobic effect.††