Change in environment of the P1 side chain upon progression from the Michaelis complex to the covalent selpin-proteinase complex

Serpins inhibit proteinases by forming a kinetically trapped intermediate during a suicide substrate inhibition reaction. To determine whether the kinetic trap involves a repositioning of the P1 side chain of the serpin following formation of the initial Michaelis complex, we used the tryptophan of a P1 M --> W variant of human alpha(1)-proteinase inhibitor as a fluorescent reporter group of the environment of the PI side chain. The P1W variant was a valid model serpin and formed SDS-stable complexes with both trypsin and chymotrypsin with a stoichiometry of inhibition close to 1.0. Rates of inhibition of chymotrypsin for wild-type and variant alpha(1)-proteinase inhibitor differred only similar to 1.8-fold. Rates of inhibition of trypsin were, however, 25-fold lower for the variant than for the wild-type inhibitor. Steady-state fluorescence spectra showed a change in environment for the P1 side chain upon forming both covalent complex with trypsin or chymotrypsin and noncovalent complex with anhydrochymotrypsin. The P1 environments in the chymotrypsin and anhydrochymotrypsin complexes were, however, different. Fluorescence quenching studies confirmed the burial of the P1 side chain upon formation of both the noncovalent and covalent complexes, but were not able to discriminate between the solvent accessibility in these complexes. Stopped-flow fluorescence measurements resolved the covalent intramolecular reaction that led to covalent complex and showed that, during the course of the covalent reaction, the environment of the P1 side chain changed consistent with a repositioning relative to residues of the proteinase active site as part of formation of the trap. This repositioning is likely to be a crucial part of the trapping mechanism.