Studies on simultaneous inhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active
against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of
their structure, interactions, function and evolution. Examination of the known three-dimensional
structures of BBIs revealed similarities and subtle differences. The hydrophobic core,
deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem
structural domains of the double headed BBI are related by an almost exact two-fold, in contrast
to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the
orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the
rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and
chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor
short contacts between the two enzymes bound to the inhibitor suggesting near independence of
binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is
associated with a minor negative cooperativity. In order to assess the functional significance of
the reported oligomeric forms of BBI, binding of proteases to the crystallographic and
non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined.
It was found that all the active sites in these oligomers cannot simultaneously participate in
inhibition.