Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities.
The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.
Boopathy
R
R
Balasubramanian
A S
AS
D015077:2-Hydroxy-5-nitrobenzyl Bromide; D000089:Acetic Anhydrides; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D001974:Bromosuccinimide; D002091:Butyrylcholinesterase / Q000097:blood* / Q000502:physiology; D002794:Choline / Q000031:analogs & derivatives; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002802:Cholinesterases / Q000097:blood*; D004047:Diethyl Pyrocarbonate; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines; D007093:Imidazoles; D007099:Imipramine; D010422:Pentanones; D010640:Phenothiazines; D010658:Phenylglyoxal; D013379:Substrate Specificity; D013774:Tetranitromethane; D014302:Trinitrobenzenesulfonic Acid
D000089:Acetic Anhydrides; D002800:Cholinesterase Inhibitors; D006898:Hydroxylamines; D007093:Imidazoles; D010422:Pentanones; D010640:Phenothiazines; C031800:acetic anhydride; D019811:Hydroxylamine; C017100:butyrylcholine; C008790:acetylacetone; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014302:Trinitrobenzenesulfonic Acid; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D000581:Amidohydrolases; C019732:aryl acylamidase; D013774:Tetranitromethane; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D002794:Choline; D007099:Imipramine; C012160:N-acetylimidazole