Synthesis and evaluation of 19F labeled sulfonyl fluorides as probes of protease structure: alpha-chymotrypsin.

Active site Ser-195-fluorine-labeled derivatives of alpha-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new 19F spin labels varied in the position of the -CF3 substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of "tosyl-chymotrypsin" were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.