molecules Review Why is Aged Acetylcholinesterase So Difficult to Reactivate? Daniel M. Quinn *, Joseph Topczewski, Nilanthi Yasapala and Alexander Lodge Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA; [email protected] (J.T.); [email protected] (N.Y.); [email protected] (A.L.) * Correspondence: [email protected]; Tel.: +1-319-335-1335 Received: 1 August 2017; Accepted: 29 August 2017; Published: 4 September 2017 Abstract: Organophosphorus agents are potent inhibitors of acetylcholinesterase. Inhibition involves successive chemical events. The first is phosphylation of the active site serine to produce a neutral adduct, which is a close structural analog of the acylation transition state. This adduct is unreactive toward spontaneous hydrolysis, but in many cases can be reactivated by nucleophilic medicinal agents, such as oximes. However, the initial phosphylation reaction may be followed by a dealkylation reaction of the incipient adduct. This reaction is called aging and produces an anionic phosphyl adduct with acetylcholinesterase that is refractory to reactivation. This review considers why the anionic aged adduct is unreactive toward nucleophiles. An alternate approach is to realkylate the aged adduct, which would render the adduct reactivatable with oxime nucleophiles. However, this approach confronts a considerable—and perhaps intractable—challenge: the aged adduct is a close analog of the deacylation transition state. Consequently, the evolutionary mechanisms that have led to transition state stabilization in acetylcholinesterase catalysis are discussed herein, as are the challenges that they present to reactivation of aged acetylcholinesterase. Keywords: acetylcholinesterase; organophosphorus agent; inhibition; reactivation; aging; transition state 1. Introduction Figure1 outlines the various chemical reactions that ensue when acetylcholinesterase (AChE) is exposed to organophosphorus (OP) inhibitors. These reactions are of considerable medicinal and national security interest, since OP agents that inhibit AChE (such as sarin in Figure1) are acute neurotoxins. Events in recent years underscore this concern: Iraq used tabun against Iranian troops in the Iran–Iraq war [1], terrorists have used sarin against civilians [2], tyrants have used sarin to mass murder their own people [3], and a dictator ordered the execution of his own brother with VX [4]. The seminal chemical reaction between sarin and AChE in Figure1 is attack of the active site serine at phosphorus with concomitant displacement of the fluoride leaving group. This initial neutral adduct is an analog of the transition states in the acylation stage of AChE catalysis [5], and its production is accelerated by the catalytic machinery of the active site; i.e., the Ser-His-Glu catalytic triad, the oxyanion hole, and the acyl binding site [6,7]. The initial adduct is trenchantly unreactive toward spontaneous hydrolysis (cf. Figure1, Nu: = H 2O), a reaction that therefore is of no medicinal import. The lack of hydrolytic reactivity of the initial adduct is rationalized herein in terms of the resemblance of the adduct to the transition states of the acylation stage of AChE catalysis. However, judiciously designed oxime nucleophiles can readily dephosphylate the initial adducts, as shown in Figure1 (Nu: = ArCH=NOH). 2-Pyridinealdoxime methiodide (2-PAM) is a component of the FDA approved standard countermeasure that is in use in the United States [8]. Unfortunately, the initial neutral adduct may convert to a monoanionic aged adduct via a dealkylation reaction, as shown in Figure1. This aging reaction is accelerated by cation-π interaction between Trp86 of the active site and the Molecules 2017, 22, 1464; doi:10.3390/molecules22091464 www.mdpi.com/journal/molecules Molecules 2017, 22, 1464 2 of 6 and the alkyl fragment that departs in the carbocationic transition state [9]. The aged adduct is remarkablyalkyl fragment unreactive. that departs Despite in the considerable carbocationic effort transitions over statethe last [9]. two The generations, aged adduct no is remarkablypracticable meansunreactive. for reactivating Despite considerable the aged adduct efforts have over been the last found, two and generations, therefore nono practicablemedicinal agents means are for availablereactivating for the aged aged AChE. adduct Why have is beenaged found,AChE andso diff thereforeicult to noreactivate? medicinal The agents answers are available to this question for aged AChE.are framed Why in is the aged following AChE so passages difficult in to reactivate?terms of the The structural answers and to thisenergetic question features are framed of the inaged the AChEfollowing adduct, passages and inwill terms hopefully of the structuralinform future and effo energeticrts to featuressolve the of knotty the aged problem AChE that adduct, reactivation and will ofhopefully aged AChE inform poses. future efforts to solve the knotty problem that reactivation of aged AChE poses. His447 His447 Ser203 Ser203 H+ + F- O- H NNHO O- H NN O Glu334 Glu334 CH3 O O (CH3)2CHO P CH3 (CH ) CHO P F 3 2 O O Neutral Adduct Reactivation H2O (CH3)2CHOH His447 H+ + Q His447 Ser203 Ser203 O- H NNH O - Glu334 O H NN+ H O Glu334 O - O O P CH3 - O O Aged Adduct Nu = H2O Q = (CH3)2CHO P CH3 O O N CHAr Nu = Oxime Q = (CH3)2CHO P CH3 O Figure 1. InhibitionInhibition of of AChE AChE by by the orga organophosphorusnophosphorus nerve agent sarin. 2. Discussion Consideration of the selective pressure that guidedguided the evolution of the catalytic power of AChE provides a framework for understanding the hydrolytichydrolytic stability of the initial phosphyl adduct and the remarkable unreactivity of aged AChE. The physiologicalphysiological context in which AChE operates is cholinergic neurotransmission in the centralcentral andand peripheralperipheral nervousnervous systems.systems. Since cholinergic neurotransmission occurs occurs on on a a millis millisecondecond to to second second time time scale, scale, it itis isapparent apparent that that the the evolution evolution of theof the catalytic catalytic power power of AChE of AChE has has been been beset beset with with the the“need “need for forspeed”. speed”. Indeed, Indeed, AChE AChE is among is among the mostthe most potent potent of biocatalysts of biocatalysts that accelerate that accelerate hydrol hydrolysisysis reactions. reactions. The second-order The second-order rate constant rate constant kcat/Km exceeds 109 M−1·s−1 at9 low−1 ionic−1 strength [10], while the turnover number kcat > 104 s−1 [11]. For4 k−cat1 /Km, kcat/Km exceeds 10 M ·s at low ionic strength [10], while the turnover number kcat > 10 s [11]. the rate constant is prominently diffusion controlled; i.e., the enzyme is functioning at the “speed For kcat/Km, the rate constant is prominently diffusion controlled; i.e., the enzyme is functioning at the “speedlimit” of limit” biological of biological catalysis catalysis [12]. [A12 ].yet A more yet more telling telling analysis analysis arises arises from from consideration consideration of of rate constants in the thermodynamic cycle of Figure2 2.. TheThe cyclecycle isis numericallynumerically informedinformed byby thethe catalyticcatalytic constants of AChE catalysis, and by Wolfenden’s determinationdetermination of the rate constant for nonenzymic neutral hydrolysis of acetylcholine, kun = 7.2 × 10−9 −s9−1 [13].−1 These values allow one to calculate the neutral hydrolysis of acetylcholine, kun = 7.2 × 10 s [13]. These values allow one to calculate catalytic acceleration effected by the enzyme when substrate concentration is << Km, a ratio that the catalytic acceleration effected by the enzyme when substrate concentration is << Km, a ratio that Wolfenden callscalls thethe catalyticcatalytic proficiencyproficiency ofof thethe enzymeenzyme [[14]:14]: k k catK (1) Catalytic Proficiency = Km =1.4×1017 M−1 Catalytic Proficiency = k = 1.4 × 10 M (1) kun The reciprocal of the catalytic proficiency is the thermodynamic dissociation constant of the acylation transition state, KTS, from which one can calculate the free energy of dissociation of the acylation transition state at T = 298 K, as in Equation (2): Molecules 2017, 22, 1464 3 of 6 The reciprocal of the catalytic proficiency is the thermodynamic dissociation constant of the acylation transition state, KTS, from which one can calculate the free energy of dissociation of the acylation transition state at T = 298 K, as in Equation (2): DGTS = −RTlnKTS = 98 kJ/mol (23 kcal/mol) (2) Molecules 2017, 22, 1464 3 of 6 This analysis shows that, for AChE, and indeed for any enzyme catalyzed reaction, catalytic power must derive from transition state stabilization. For AChE, the acylation transition state is 98 kJ/mol ΔG = −RTlnK =98kJ/mol(23kcal/mol) (2) more stable than the transition state of the spontaneous hydrolysis of acetylcholine, an observation This analysis shows that, for AChE, and indeed for any enzyme catalyzed reaction, catalytic that can be interpreted in terms of the elements of molecular recognition that the enzyme brings to power must derive from transition state stabilization. For AChE, the acylation transition state is 98 kJ/mol bear on themore acylation stable than transition the transition state state [6]. of Athe similar spontaneous analysis hydrolysis can beof acetylcholine, considered an when observation AChE operates under conditionsthat can be of interpreted substrate in saturation; terms of the i.e.,elements kcat ofis molecular rate limiting: