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 intermedi ate during a suicide substrate inhibition reaction. To determine wheth er the kinetic trap involves a repositioning of the P1 side chain of t he serpin following formation of the initial Michaelis complex, we use d 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-s table complexes with both trypsin and chymotrypsin with a stoichiometr y of inhibition close to 1.0. Rates of inhibition of chymotrypsin for wild-type and variant alpha(1)-proteinase inhibitor differred only sim ilar to 1.8-fold. Rates of inhibition of trypsin were, however, 25-fol d lower for the variant than for the wild-type inhibitor. Steady-state fluorescence spectra showed a change in environment for the P1 side c hain upon forming both covalent complex with trypsin or chymotrypsin a nd noncovalent complex with anhydrochymotrypsin. The P1 environments i n the chymotrypsin and anhydrochymotrypsin complexes were, however, di fferent. Fluorescence quenching studies confirmed the burial of the P1 side chain upon formation of both the noncovalent and covalent comple xes, but were not able to discriminate between the solvent accessibili ty 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 environm ent of the P1 side chain changed consistent with a repositioning relat ive 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 tra pping mechanism.