Single peptide bond hydrolysis/resynthesis in squash inhibitors of serine proteinases. 1. Kinetics and thermodynamics of the interaction between squash inhibitors and bovine beta-trypsin.

The substrate and inhibitory parameters are described for the interaction between Cucurbita maxima trypsin inhibitor I (CMTI I) and bovine beta-trypsin. The data are fully consistent with the reactive site hypothesis and the standard mechanism proposed for the protein inhibitor-serine proteinase interaction. The second-order association rate constant (k(on)) for the interaction of the intact inhibitor and trypsin is high, above 10(6) M-1 s-1. The same value is only 22-fold lower for the reactive site hydrolyzed inhibitor. This result implicates a very low transition-state barrier for the hydrolysis of the Arg5-Ile6 reactive site peptide bond. The equilibrium constant Ka (= 1/Km,f) and K(assoc) change by 6 orders of magnitude in the pH range 4.0-8.3. The steady-state parameters for the hydrolysis and resynthesis of the reactive site have been determined over the pH range 3.2-8.3. Catalytic rate constants, but not kcat/km, exhibit strong pH dependence. The dependence of the hydrolysis constant (Khyd) on pH fits the simplest form of the Dobry equation, indicating that after the hydrolysis of the reactive site, pK values of any preexistent groups are not perturbed. It is suggested that a major factor leading to high kcat/Km values is the presence of Arg or Lys residues at the P1 position. Low values of Km result from a conservation of the ground-state conformation of the inhibitor binding loop upon the complex formation. The crucial stage of the reactive site hydrolysis seems to be associated with a change of basic side-chain interactions within the S1 binding pocket.