13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

NMR studies of the complex between trypsin and soybean trypsin inhibitor with 1-¹³C-arginine and modified inhibitor with 1-¹³C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsin/¹³C-Lys-inhibitor at pH 6.5 and 8.1 and for trypsin/¹³C-Arg-inhibitor at pH 5.0. At pH 7.1 for trypsin/¹³C-Arg-inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor.     

The structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor

The structure of the complex between anhydro-trypsin and pancreatic trypsin inhibitor has been determined by difference Fourier techniques using phases obtained from the native complex (Huber et al., 1974). It was refined independently by constrained crystallographic refinement at 1.9 å resolution. The anhydro-complex has Ser 195 converted to dehydro-alanine. There were no other significant structural changes. In particular, the high degree of pyramidalization of the C atom of Lys 15 (I) of the inhibitor component observed in the native complex is maintained in the anhydro-species.     

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.