Binding of porcine pancreatic secretory trypsin inhibitor to bovine beta-trypsin: a kinetic study
Ascenzi, P.; Amiconi, G.; Bolognesi, M.; Menegatti, E.; Guarneri, M.
Biopolymers 25(12): 2325-2333
The kinetics of the formation of the complex between bovine .beta.-trypsin and the porcine pancreatic secretory trypsin inhibitor (PSTI; Kazal-type inhibitor) was investigated following the spectral changes associated with the displacement of proflavine from the enzyme, upon inhibitor binding, between pH 3.5 and 8.0 (I = 0.1M) at 21 .+-. 0.5.degree. C. With inhibitor in excess over the enzyme ([PSTI] .gtoreq. 5 .times. [bovine .beta.-trypsin]), the time course of the reaction corresponds to a pseudo-first-order process. Over the whole pH range explored, the concentration dependence of the rate is second order at low PSTI concentrations but tends to first order at high inhibitor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting first-order process. Values of kinetic parameters for PSTI binding to bovine .beta.-trypsin depend, between pH 3.5 and 8.0, on the acid-base equilibrium of a single ionizing group (probably His-57 of bovine .beta.-trypsin) that undergoes an acidic pKa shift from 7.0 in the free bovine .beta.-trypsin to 5.5 in the enzyme:PSTI complex. Kinetics of the bovine .beta.-trypsin:PSTI adduct formation has been analyzed and compared with that of other (pro)enzyme:inhibitor reactions. Considering the known molecular structures of free serine (pro)enzymes, of Kazal- and Kunitz-type inhibitors, as well as of their complexes, the binding behavior of PSTI to bovine .beta.-trypsin has been related to the inferred stereochemistry of the proteinase:inhibitor contact region.