ABSTRACT
Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min-1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53400 min-1). With a kcat/Km = (2.6 ± 1.6) × 107 M-1min-1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.
Subject(s)
Biocatalysis , Echothiophate Iodide/metabolism , Enzymes/metabolism , Organophosphorus Compounds/metabolism , Spectrometry, Fluorescence , Butyrylcholinesterase/metabolism , Caulobacteraceae/enzymology , Echothiophate Iodide/chemistry , Humans , Hydrolysis , Kinetics , Molecular Docking Simulation , Mutant Proteins/metabolism , Phosphoric Triester Hydrolases/metabolism , Sulfolobus/enzymologyABSTRACT
The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.
Subject(s)
Butyrylcholinesterase/chemistry , Cholinesterase Inhibitors/chemistry , Echothiophate Iodide/chemistry , Isoflurophate/chemistry , Paraoxon/chemistry , Biocatalysis , Butyrylcholinesterase/genetics , Butyrylcholinesterase/metabolism , Catalytic Domain , Cholinesterase Inhibitors/metabolism , Echothiophate Iodide/metabolism , Hydrolysis , Isoflurophate/metabolism , Kinetics , Molecular Dynamics Simulation , Mutation , Paraoxon/metabolism , Protein Binding , Protein Engineering , ThermodynamicsABSTRACT
Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.
Subject(s)
Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Organophosphorus Compounds/metabolism , Acetylthiocholine/metabolism , Acetylthiocholine/pharmacology , Animals , Bungarus , Chlorpyrifos/analogs & derivatives , Chlorpyrifos/pharmacology , Disulfoton/pharmacology , Echothiophate Iodide/metabolism , Echothiophate Iodide/pharmacology , Isoflurophate/metabolism , Isoflurophate/pharmacology , Mutagenesis, Site-Directed , Mutation , Paraoxon/metabolism , Paraoxon/pharmacologyABSTRACT
The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.
Subject(s)
Butyrylcholinesterase/genetics , Butyrylcholinesterase/metabolism , Esterases/genetics , Esterases/metabolism , Animals , Aryldialkylphosphatase , Base Sequence , Butyrylcholinesterase/chemistry , CHO Cells , Cricetinae , DNA/genetics , Echothiophate Iodide/chemistry , Echothiophate Iodide/metabolism , Esterases/chemistry , Humans , In Vitro Techniques , Insecticides/chemistry , Insecticides/metabolism , Kinetics , Miotics/chemistry , Miotics/metabolism , Molecular Structure , Mutagenesis, Site-Directed , Paraoxon/chemistry , Paraoxon/metabolism , Point Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Human pancreas contains two cholinesterase isoenzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In the present study, binding potency of two organophosphates for human cholinesterases were compared by the Ellman method. Echothiophate was found to have much greater potency than iso-OMPA for both cholinesterases. Using Karnovsky histochemical stains on human pancreatic tissue, the same results were confirmed. Dose-response studies with acetylcholine were done on viable pancreas fragments from nine human donors, without pancreatic disease (group I). Cold-preservation time was less than 30 h. Pancreas was minced into fragments, after the technique of Scheele and Palade, placed in Eagle's medium, and gassed with O2. Amylase release was measured by the Phadebas Method and corrected for basal release. There was a dose-dependent response to acetylcholine at 1 and 2 h, with a shift in peak amylase release to the left, when fragments were preincubated in 10(-4) M echothiophate. This indicated a 100-fold increase in sensitivity to acetylcholine. In three patients with chronic pancreatitis (Group II), there were variable patterns of response of amylase release to acetylcholine, and higher basal outputs. In Group III, prolonged storage conditions of over 40 h were tested for 4 pancreas donor tissues. There was no response to acetylcholine. These studies show that for up to 30 h cold storage, fragments of pancreas from human organ donors respond to acetylcholine in dose-dependent manner. An organophosphate, echothiophate (10(-4) M) which inhibits both cholinesterases, increases pancreatic sensitivity to acetylcholine, and these results are similar to findings from canine pancreas fragments, which also showed increased sensitivity.
Subject(s)
Acetylcholine/pharmacology , Organophosphorus Compounds/pharmacology , Pancreas/drug effects , Amylases/metabolism , Cholinesterases/metabolism , Dose-Response Relationship, Drug , Echothiophate Iodide/metabolism , Echothiophate Iodide/pharmacology , Humans , Organophosphorus Compounds/metabolism , Pancreas/enzymology , Pancreas/metabolism , Pancreatitis/chemically induced , Tetraisopropylpyrophosphamide/metabolism , Tetraisopropylpyrophosphamide/pharmacologyABSTRACT
A 72-year-old man with epiphora secondary to bilateral canalicular stenosis resulting from long-term treatment with 0.125% to 0.25% echothiophate iodide (phospholine iodide) drops for glaucoma underwent bilateral conjunctivodacryocystorhinostomies with Jones' tubes. Within days after undergoing this surgery, he experienced severe unexplained diarrhea, fatigue, weight loss, and prostration. He cancelled his postoperative ophthalmic appointment because of "medical illness." He required admission to his local hospital where extensive studies were done in an attempt to establish the cause of this life-threatening condition. After stopping the echothiophate iodide drops, all symptoms disappeared within two days. Drug toxicity is a previously unreported complication of conjunctivodacryocystorhinostomy, and this case demonstrates that topical medications have enhanced systemic absorption after lacrimal surgery with placement of fistulizing prosthetic devices. One must be aware of this possible complication, not only with long-acting anticholinesterases, but with topical sympathomimetic drugs (especially in cardiac patients) as well as cycloplegic agents in children.
Subject(s)
Dacryocystorhinostomy , Echothiophate Iodide/adverse effects , Intubation/adverse effects , Aged , Conjunctiva/surgery , Diarrhea/chemically induced , Echothiophate Iodide/metabolism , Humans , Lacrimal Apparatus/surgery , Lacrimal Duct Obstruction/chemically induced , Male , Nasal Cavity/surgerySubject(s)
Echothiophate Iodide/metabolism , Lens, Crystalline/metabolism , Animals , Aqueous Humor/analysis , Aqueous Humor/metabolism , Cornea/analysis , Cornea/metabolism , Echothiophate Iodide/analysis , Echothiophate Iodide/blood , In Vitro Techniques , Injections , Iris/analysis , Iris/metabolism , Lens, Crystalline/analysis , Rabbits , Radiometry , Retina/analysis , Retina/metabolism , Sclera/analysis , Sclera/metabolism , Tritium , Vitreous Body/analysis , Vitreous Body/metabolismABSTRACT
The tertiary analogute of phospholine, namely, (C(2)H(5)O)(2)P(O)SCH(2)CH(2)N(CH(3))(2), is a potent, irreversible inhibitor of cholinesterase which, when externally applied to the sqluid giant axon, readily penetrates in its inhibitory form into the axoplasm. However, even a 10(-2) molar solution of this compound does not block axonal conduction unless the axon is first treated with a low concentration of venom from the cottonmouth moccasin. The question of the activity of acetylcholinesterase in these axons is considered, and the possibility of subcellular permeability barriers for indivisual components of the excitable membrane is discussed.