Detailed Mechanism of Interaction of Bovine β-Trypsin with Soybean Trypsin Inhibitor (Kunitz)

Stopped-flow kinetics of the resynthesis of the reactive site peptide bond in kallikrein inhibitor (Kunitz) by β-trypsin

“…Most likely I* and enzyme first form a more labile complex L* at a diffusioncontrolled rate. A complex L* with a similar stability as L was indeed observed for the reaction of modified soybean inhibitor with /3-trypsin (Luthy et al, 1973). L* is therefore tentatively included in the following scheme which summarizes the rate constants at pH 7.5 and at room temperature:…”

“…The fact that the amplitudes of the two phases in Figure 1 are of comparable magnitude whereas the respective half-times differ by more than a factor of 100 proves that k-x is much larger than kc (for a detailed discussion, see Quasi et al, 1974;Luthy et al, 1973). Therefore the intermediate X is in equi- Slow build-up of an absorption difference spectrum following mixing of 3-trypsin with I*.…”

“…When virgin kallikrein inhibitor or virgin soybean inhibitor react with /3-trypsin (Luthy et al, 1973;Finkenstadt et al, 1974;Quasi et al, 1976) or with -chymotrypsin (Quasi et al, 1974), the formation of a Michaelis complex L precedes the formation of the final stable complex C. This first encounter is too fast to be resolved by stopped-flow techniques and it is probably a nearly diffusion-controlled reaction (Quasi et al, 1974;Engel et al, 1974). The intermediate X which was demonstrated for the reaction of modified kallikrein inhibitor 1* with /3-trypsin in the present study is formed 50 to 1000 times more slowly and is about 100 times more stable than L. Therefore X is not the encounter complex.…”

“…In this case complex formation is accompanied by a resynthesis of the peptide bond and must proceed via another sequence of catalytic steps including the formation of the acyl intermediate. So far pertinent results are available only for the reaction of modified soybean trypsin inhibitor with /3-trypsin (Luthy et al, 1973). Some preliminary data on the interaction of /3-trypsin with the much smaller and chemically different trypsin kallikrein inhibitor in its modified form were presented by Quasi et al (1975,1976).…”

Lattice Interactions in Crystals of Soybean Trypsin Inhibitor (Kunitz) Produced by Inclusion of 1,5-Disulfonylnaphthalene

“…These are designated Kunitz type inhibitors, after Moses Kunitz, who first discovered, purified, and crystallized them, and Bowman–Birk inhibitors, named after their discoverers. From a biological standpoint, the trypsin inhibitors are of significance because of their nutritional and antinutritional properties, because of their contribution to plant defense, and for what they reveal about the mechanisms of inhibition of various proteases. − They have also been used in the treatment of a number of human diseases, including cancer, where inhibition of protease activity slowed progression …”