Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes.
The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.
D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D004735:Energy Transfer; D046911:Macromolecular Substances; D010940:Plant Proteins; D011480:Protease Inhibitors; D011485:Protein Binding; D011487:Protein Conformation; D012639:Seeds / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014357:Trypsin; D014361:Trypsin Inhibitors
D046911:Macromolecular Substances; D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin