Molecular Recognition in Acetylcholinesterase Catalysis: Free-Energy Correlations for Substrate Turnover and Inhibition by Trifluoro Ketone Transition-State Analogs

Self Cite

212

248

To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of ؉4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK ؉ ) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK ؉ reaction with the active center but inverts the ionic strength dependence for the cationic TFK ؉ association with AChE. Hence it appears that TFK ؉ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of ؉4.

Section: Effect Of Ionic Strength On Rate Constants For Substrate Andmentioning

confidence: 99%

Electrostatic Influence on the Kinetics of Ligand Binding to Acetylcholinesterase

Radić¹,

Kirchhoff

Quinn

et al. 1997

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248

“…This difference suggests that the orientation of this hemiketal conjugate differs where the t-butyl group extends toward the gorge exit. TFK 0 inhibits the wild type enzyme 70-fold slower than TFK ϩ , presumably due to lack of cation-interaction and slightly different ligand orientation (21). Alkyl phosphorylation with small alkyl groups also has little influence on the environment at position 124 (Table IV).…”

Section: Influence Of Residue Modification On Ligand Binding-mentioning

confidence: 99%

Reversibly Bound and Covalently Attached Ligands Induce Conformational Changes in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Shi

Boyd²,

Radić

et al. 2001

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu 76 , Glu 81 , Glu 84 , Tyr 124 , Ala 262 , and His 287 ) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (⍀ loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the ⍀ loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the ⍀ loop accompany ligand binding.Acetylcholinesterase (AChE), 1 a serine hydrolase in the ␣/␤-fold hydrolase protein superfamily (1), terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholine at a diffusion limited rate (2, 3). The crystallographic structure of mouse AChE reveals a catalytic triad (Ser 203 , Glu 334 , and His 447 ) located at the bottom of a narrow active site gorge 20 Å in depth (4 -6). Because the cross-section of the physiological substrate acetylcholine is larger than the narrowest part of the gorge, the remarkably high turnover rate of AChE raises questions regarding substrate access to the catalytic site.Molecular dynamic simulations suggest that rapid fluctuations of gorge width combined with diffusion facilitated by electrostatic forces could enhance substrate accessibility (7-10). In addition, the high affinity and slowly dissociating complex of fasciculin and AChE retains slight residual catalytic activity (11, 12), despite the occlusion of the active site gorge by fasciculin as shown in the crystal structures (5, 13, 14). Rapid fluctuations in residues lining the gorge walls may leave transient gaps at the fasciculin-AChE interface and may account for residual activity.The large omega loop (⍀ loop), Cys 69 -Cys 96 , flanking the active site gorge in mouse AChE corresponds to the activation loop ...

“…Reactivity toward the Transition State Analog TMTFA-The kinetics of AChE inhibition by TMTFA has been studied extensively since the tetrahedral covalent adduct is believed to mimic the transition state of the acylation process (21,28,29). TMTFA was shown to behave as a tight binding time-dependent inhibitor, a behavior characteristic of other trifluoroketone inhibitors of serine proteases (30,31).…”

Section: Modification Of Huache Hydrolytic Activity-replacementmentioning

confidence: 99%

Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase

Ordentlich¹,

Barak²,

Kronman³

et al. 1998

149

The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000 -11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000 -6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500 -460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket.