Substrate Report for: O-Nitrophenylbutyrate

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect 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-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl 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 perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect 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-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl 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 perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.