The indirect mechanism of action of the trifluoroacetyl peptides on elastase. Enzymatic and 19F NMR studies.

Trifluoroacetyl (CF3CO) dipeptide anilides are potent reversible inhibitors of elastase. Their 19F NMR spectra in the presence of the enzyme correspond therefore to slow chemical exchange. Their characteristics are very similar to those previously reported for other CF3CO-peptides. This should correspond to a single binding mode, in which the CF3CO group lies in a specific site in close contact with protein protons. Elastase, irreversibly inhibited by alkylation of Ser-195 with phenylsulfonyl fluoride derivatives, binds the CF3CO-Ala-containing dipeptide anilides still more tightly than the native enzyme. It no longer binds the dipeptide anilides containing a bulky CF3CO-Lys group, suggesting a location of the CF3CO site near the S1 subsite. On the other hand, the chemical shift of the CF3CO 19F resonance in the complex is the only NMR property affected by the enzyme sulfonylation, the T1 and nuclear Overhauser effect values being unmodified as compared to those in the complexes with the native enzyme. The observation of the NMR characteristics of elastases inhibited by phenylsulfonyl groups substituted with fluorine shows that complexation with CF3CO-peptides induces a change of conformation of the catalytic site which should correspond to a displacement of His-57 toward the ortho position of the phenylsulfonyl ring. Such a transconformation is not observed with corresponding acetylated peptides. The phenylsulfonyl fluoride derivatives are still able to react with elastase in the presence of a large excess of CF3CO-peptides. In such conditions the rate of inactivation is much slower but still at least 5% of that measured in the absence of the reversible inhibitors. This residual activity is hardly compatible with the presence of two exclusive modes of interaction of the CF3CO-peptides with native elastase. On the contrary, these observations are better interpreted by a single and identical mode of binding of the peptide to the native and sulfonylated enzymes. According to this mode of binding, the reversible inhibition of elastase by CF3CO-peptides should correspond to an indirect mechanism by which a change of conformation at the active site results in a reduced catalytic activity.