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Abstract
The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0°C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH8.0, values of the apparent ther-modynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: K, = 4.9 × 104M-1, ΔG° = -6.3kcal/mol, and ΔS° = +37 entropy units (all at 21.0°C); and ΔH° = + 4.6 kcal/mol (temperature independent over the explored range, from 5.0°C to 45.0°C).
Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme/Kazal-and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.