A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine

Abstract

β-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L₃-thiocholine (L = H or ²H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis–Menten kinetics, and yield the following isotope effects: ^D3k_cat/K_m = 0.98 ± 0.02 and ^D3k_cat = 1.10 ± 0.02. The modestly inverse isotope effect on k_cat/K_m is consistent with partial rate limitation by a step that converts the sp²-hybridized ester carbonyl of the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k_cat indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538–14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.

Introduction

The cholinesterases comprise a family of enzymes that are noted for their catalytic power [1], [2]. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine and acetylthiocholine (ATCh) with rate constants that approach diffusion control at low substrate concentrations, and accelerates the hydrolysis of acetylcholine by 10¹³-fold. Though an order of magnitude slower than AChE, BuChE nonetheless effects catalytic turnover of ATCh and butyrylthiocholine (BuTCh) with admirable efficiency [3]. Elucidating the molecular origins of the high catalytic activity of cholinesterases is therefore an endeavor of fundamental importance. However, due to the very high catalytic activities of cholinesterases (k_cat ∼ 10⁴ s⁻¹ for AChE-catalyzed hydrolysis of ATCh [1], [2]; k_cat ∼ 10³ s⁻¹ for BuChE-catalyzed hydrolysis of BuTCh [3]), characterization of the structural changes in the substrate that accompany catalytic turnover can be challenging.

Kinetic secondary isotope effects provide probes of chemical reaction mechanisms, including those that occur in the active sites of enzymes that report on structural changes as the substrate is converted from the reactant state to the transition state. Consider, for example, the conversion of acetylthiocholine to a tetrahedral intermediate (one of the several steps in acyl transfer reactions [4]) in the acylation stage of catalysis, as outlined in Fig. 1. Nucleophilic attack of the active site serine on the substrate converts the carbonyl carbon from sp² hybridization in the reactant state to sp³ hybridization in the tetrahedral intermediate. In the reactant state, the carbonyl CO has a marked bond dipole that is δ⁺ at the carbonyl carbon. This and the π-system of CO allow for resonance delocalization of β-CL (L = H or ²H) σ-electron density into the carbonyl system by hyperconjugation [5]. This electron delocalization decreases electron density in the β-CL bonds. In the tetrahedral intermediate the π-system is no longer available, hyperconjugation is not possible, and electron density is returned to β-CL bonds, which increases the strength (stiffness) of the bonds. The increased stiffness of β-CL bonds in the tetrahedral intermediate increases the curvature of the vibrational potential in the tetrahedral intermediate versus the reactant state, as shown in Fig. 2. This renders the isotopic difference in β-CL zero point energies larger in the tetrahedral intermediate than in the reactant state, which in turn necessitates that ΔE^H3 > ΔE^D3, and therefore ^D3K≡K_H3/K_D3 = 0.8. Correspondingly, the kinetic β-secondary deuterium isotope effect ^D3k≡k_H3/k_D3 will be increasingly inverse (i.e. will more closely approach the equilibrium isotope effect ^D3K = 0.8) the more the transition state resembles the tetrahedral intermediate. Therefore, inverse kinetic β-secondary deuterium isotope effects for acyl transfer reactions not only indicate that the sp²-hybridized reactant state is being converted to a sp³-hybridized tetrahedral intermediate, but also provide a measure of how far the reaction assembly has progressed from sp² to sp³ hybridization in the transition state [5], [6].

When the substrate in the reactant state is sp³ hybridized and hybridization is changing toward sp² in the transition state, the situation is the reverse of that depicted in Fig. 2. In this case the β-CL vibrational potential will be shallower in the transition state than in the reactant state, and the kinetic β-secondary deuterium isotope effect will be normal, i.e. ^D3k > 1.0. Moreover, the upper limit for ^D3k, i.e. that for equilibrium conversion of the sp³ reactant state to the sp² product should be 1/0.8, or about 1.25.

What are the expectations for kinetic β-secondary deuterium isotope effects for acyl transfer reactions, of which the elementary step shown is Fig. 1 is a component? Most acyl transfer reactions proceed through addition–decomposition mechanisms, in which the nucleophile first attacks the susceptible carbonyl carbon to generate a tetrahedral intermediate, which breaks down with the expulsion of the leaving group to produce the products [4]. Tetrahedral intermediates in nonenzymic acyl transfer reactions are high-energy, steady-state intermediates, less stable than reactants or products by 40–100 kJ mol⁻¹[4]. Therefore, according to the Hammond postulate [7] the transition states for formation and breakdown of the tetrahedral intermediate should resemble the intermediate both in energy and structure. In this situation, kinetic β-secondary deuterium isotope effects will always be inverse, no matter whether formation or breakdown of the intermediate is rate limiting, as illustrated in Fig. 1, Fig. 2.

Enzymes achieve their impressive catalytic power, as measured by the degree to which they accelerate erstwhile sluggish nonenzymic reactions, by lowering the energy of the transition state(s), which in turn lowers the free energy of activation [8]. Understanding how enzymes do this is the crux of understanding their catalytic function. A way in which cholinesterases, and other enzymes that catalyze acyl transfer reactions, may realize a substantial portion of their catalytic accelerations is to stabilize high-energy tetrahedral intermediates, which in turn will stabilize the transition states for their formation and decomposition. In this paper, results of isotope effect experiments are described that show that equine serum BuChE stabilizes a tetrahedral intermediate to such an extent that the intermediate is an accumulating Michaelis complex (i.e. reactant state) during catalytic turnover. Results for equine serum BuChE are compared to those in the literature for human BuChE catalysis [9], and are discussed in light of observations of tetrahedral intermediates in X-ray structures of the enzyme [10].