MECHANISTIC STUDY OF HLE INHIBITION USING DUAL LABELED MACROMOLECULARINHIBITOR

The mechanism of inhibition of a specific and effective (K-i = 1-10 nM ) macromolecular inhibitor of HLE was investigated. The inhibitor, pol ymer-bound peptidyl carbamate 1 was labeled with [SH] at its polymeric backbone (Mw = 27,000) and with [C-14] in it peptidyr carbamate moiet y. When the macromolecular inhibitor 1 was incubated with HLE to compl ete inhibition and then competitively displaced by an HLE substrate, o nly intact [H-3/C-14] polymer-bound inhibitor 1 was recovered. At the same time complete restoration of enzymatic activity was achieved. Gel permeation chromatography and HPLC were utilized to eliminate the pos sibility of the presence of low molecular weight fragments resulting f rom the interaction of 1 with HLE. It is concluded that 1 exerts its i nhibitory action on HLE without the prior release of the low molecular weight inhibitor 3 (Mw = 570).