Reversible and competitive inhibition of enteropeptidase by 1-trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64)

l-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), an well known cysteine proteinase inhibitor, which specifically inhibits cysteine proteinases by irreversibly modifying the free sulfhydryl group of the active site cysteine residue with its epoxide group, was shown to strongly inhibit the serine proteinase enteropeptidase, the key enzyme in the intestinal protein digestion cascade, in a reversible and competitive manner with a K_i value of 12 μM. The derivative of E-64, in which the epoxide group was reacted with 2-mercaptoethanol, also inhibited the enzyme, whereas 1-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), which lacks the guanidinium group, had no inhibitory effect on the enzyme, indicating that the guanidinium group is essential for the inhibition of enteropeptidase.