Enzymatic properties of alpha 2-macroglobulin-proteinase complexes. Apparent discrimination between covalently and noncovalently bound trypsin by reaction with soybean trypsin inhibitor.
The binding of trypsin to alpha 2-macroglobulin, the appearance of free beta-cysteinyl thiol groups of the formed complexes, the steady-state kinetics of their enzymic hydrolysis of carbobenzoxy-L-valyl-glycyl-L-arginyl-4-nitroanilide and finally their reactions with soybean trypsin inhibitor leading to the formation of ternary alpha 2-macroglobulin-trypsin-soybean trypsin inhibitor complexes were investigated. Each alpha 2-macroglobulin molecule binds two trypsin tightly; the dissociation constants were found to be unmeasureably small, but the extent of formation of 1:1 and 1:2 complexes at different molar ratios of alpha 2-macroglobulin to trypsin as determined from the appearance of thiol groups clearly indicated that binding of trypsin to alpha 2-macroglobulin shows negative cooperativity. Binding of the first trypsin makes the access of the second less easy. The kinetic results showed a decrease of the kc/Km value of hydrolysis of the tripeptide substrate by approx. 4-fold compared to that of free trypsin for each alpha 2-macroglobulin-bound trypsin. Here no differences were seen between the bound trypsins. The analysis of the reactions between the alpha 2-macroglobulin-trypsin complexes and soybean trypsin inhibitor shows that ternary complexes do form, although slowly, and that two processes occur, not only when 1:2 complexes but also when 1:1 complexes react with soybean trypsin inhibitor. Soybean trypsin inhibitor apparently discriminates between two distinct binding modes of trypsin to alpha 2-macroglobulin, the covalently and the noncovalently alpha 2-macroglobulin-bound trypsins.
D000818:Animals; D002417:Cattle; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism*; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D046911:Macromolecular Substances; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; D014357:Trypsin