Mechanism of inactivation of a Fasciola proteolytic enzyme by peptide aldehydes and alkylating agents

A proteolytic enzyme of the liver fluke Fasciola sp. was purified as described previously by ammonium sulfate fractionation, gel filtration on Sephadex G-200 column and l-phenylalanine-agarose chromatography. Leupeptin, a peptide aldehyde of microbial origin, competitively inhibited the enzyme activity with respect to the substrate $\text{α-N-}\text{benzoyl-}\text{l}\text{-argininamide}$; the apparent K_i value for leupeptin is 45 000-fold less than the apparent K_m for the substrate. Incubation of the enzyme with leupeptin resulted in time-dependent inactivation of the globinolytic activity, with an inactivation constant (K_inact) of 0.4 μM giving the half-maximum inactivation velocity, and with a minimum inactivation half-time (T) of 2.7 min at infinite concentration of this compound. The inactivated enzyme was not reactivated by extensive dialysis. These results imply that leupeptin yields an affinity labelling of an active site of the enzyme. The activity of the Fasciola proteolytic enzyme was also inactivated by other peptide aldehydes and alkylating agents and inactivation constants observed were 0.5 μM for chymostatin, 13 μM for antipain, 2 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-lysine}$ chloromethyl ketone, 140 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-phenylalanine}$ chloromethyl ketone and 40 μM for iodoacetate under the conditions used.