SUBSTRATE DEPENDENCE OF AMILORIDE-INDUCED AND SOMAN-INDUCED CONFORMATION CHANGES OF BUTYRYLCHOLINESTERASE AS EVIDENCED BY HIGH-PRESSURE PERTURBATION

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-ni trophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman precedi ng enzyme phophonylation induced a new enzyme conformational state (E' ). The purpose of the present study was to determine whether this effe ct depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-cat alyzed hydrolysis of nitrophenyl esters. The effect of amiloride was f ound to be dependent on the position ortho or para of the substrate ni tro group: amiloride acts as a non-linear reversible activator of p-ni trophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/p-nitrophenylbutyrate was also studied under perturbin g conditions, i.e., as a function of pressure (1-1600 bar) in the pres ence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of ami loride depends on the position of the substrate nitro group and amilor ide concentration. Molecular modelling suggests that the presence of a miloride determines the orientation of ortho- and para-nitrophenyl est ers in the active-site gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ri ng interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hol e but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acy l chain. Thus, the network of interactions in ternary complexes is tig hter with o-nitrophenylbutyrate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-n itrophenylbutyrate is the substrate. On the other hand, reversible bin ding of amiloride and/or soman induces new active conformational state s that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzy me conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.