Mechanism of Association of a Specific Aldehyde Inhibitor, Leupeptin, with Bovine Trypsin1

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Abstract
Leupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptin with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps: The observed dissociation constant (K1) for the first step was 1.24×10−3M (at pH 8.2, 15°C and the two first-order rate constants (K+2 and K−2) were 166s−1 and 1.75×10−2.s− respectively (at pH 8.2, 15°C). The dissociation constant (K2) for the whole process was calculated from these parameters to be 1.34×10−8M. This value is compatible with that determined directly by an independent static method (2.36×10−8M). We also measured Kd for the leupeptin complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A leupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay system in the active site is important for the formation and stabilization of complex II.