Hydrolysis-Resynthesis Equilibrium of the Lysine-15–Alanine-16 Peptide Bond in Bovine Trypsin Inhibitor (Kunitz)

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Abstract
Catalytic amounts of bovine .beta.-trypsin [EC 3.4.21.4], bovine .alpha.-chymotrypsin [EC 3.4.21.1] and porcine plasmin [EC 3.4.21.7] establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I*) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates for .beta.-trypsin are at pH 3.75, for .alpha.-chymotrypsin at pH 5.5 and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10-5M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. The hydrolysis constant KHyd = [I*]/[I] is 0.33 at pH 5.0. This demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase and that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.