Spin-labeled Sulfonyl Fluorides as Active Site Probes of Protease Structure

a-Chymotrypsin and trypsin were spin-labeled with 14 ortho-, meta-, and para-substituted phenylsulfonyl fluorides. These analogues of tosylfluoride were substituted with either pyrrolidinyl, pyrrolinyl, or piperidinyl nitroxide moieties. A complete description of their synthesis and the enzyme inhibition studies is presented in the following paper (WONG, S. S., QUIGGLE, K., TRIPLETT, C., AND BERLINER, L. J. (1974) J. Biol. Chem. 249, 1678-1682). The studies were designed to test for “dynamic conformational homologies” in these closely related proteins. However, the spectral results at pH 3.5 clearly showed that the active site conformations of trypsin and a-chymotrypsin were different. Model-building studies indicated which “tosyl” spin labels could bind isomorphously to tosyl-archymotrypsin. Several of those labels which were shown to be restricted structurally from binding in the chymotrypsin aromatic pocket were found upon exposure to saturated indole to give electron spin resonance spectra closely resembling those for trypsin. Those inhibitors which could theoretically bind in the pocket were affected least or not at all. With trypsin, on the other hand, 1 mM benzamidine produced no spectral shifts. A suggested model for cY-chymotrypsin based on steric arguments provides for three general binding modes: (a) one isomorphous to tosylchymotrypsin, (b) one reflecting partial binding at the tosyl hole, and (c) one in a general region of the active site. Indole can displace the partially bound State b toward the general binding Mode c. This general Mode c is probably common to trypsin as well.