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. 2009 Aug 26;131(33):11964-75.
doi: 10.1021/ja903990p.

Fundamental reaction mechanism and free energy profile for (-)-cocaine hydrolysis catalyzed by cocaine esterase

Junjun Liu  1 , Adel Hamza, Chang-Guo Zhan
Affiliations

Fundamental reaction mechanism and free energy profile for (-)-cocaine hydrolysis catalyzed by cocaine esterase

Junjun Liu et al. J Am Chem Soc. .

Abstract

The fundamental reaction mechanism of cocaine esterase (CocE)-catalyzed hydrolysis of (-)-cocaine and the corresponding free energy profile have been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations. On the basis of the QM/MM-FE results, the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by the hydroxyl group of Ser117, dissociation of (-)-cocaine benzoyl ester, nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by water, and finally dissociation between the (-)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule was found to be rate-determining, which is remarkably different from (-)-cocaine hydrolysis catalyzed by wild-type butyrylcholinesterase (BChE; where the formation of the prereactive BChE-(-)-cocaine complex is rate-determining) or its mutants containing Tyr332Gly or Tyr332Ala mutation (where the first chemical reaction step is rate-determining). Besides, the role of Asp259 in the catalytic triad of CocE does not follow the general concept of the "charge-relay system" for all serine esterases. The free energy barrier calculated for the rate-determining step of CocE-catalyzed hydrolysis of (-)-cocaine is 17.9 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 16.2 kcal/mol. In the present study, where many sodium ions are present, the effects of counterions are found to be significant in determining the free energy barrier. The finding of the significant effects of counterions on the free energy barrier may also be valuable in guiding future mechanistic studies on other charged enzymes.

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Figures

Scheme 1
Scheme 1
Proposed catalytic mechanism for CocE-catalyzed hydrolysis of (−)-cocaine where Ser117 acts as nucleophile in the first reaction step.
Scheme 2
Scheme 2
Possible catalytic mechanism for CocE-catalyzed hydrolysis of (−)-cocaine where Ser117 acts as a general base to activate a water molecule and the activated water molecule acts as a nucleophile in the first reaction step.
Figure 1
Figure 1
Division of the QM/MM systems for simulating the CocE-catalyzed (−)-cocaine hydrolysis. Atoms in blue are treated by QM method. Three boundary carbon atoms (Cα, colored in red) are treated with the improved pseudobond parameters (ref. 26). All other atoms belong to the MM subsystem.
Figure 2
Figure 2
Plots of the key internuclear distances (D1 to D4) vs the simulation time in the MD-simulated ES complex.
Figure 3
Figure 3
Plots of the key internuclear distances (D5 and D6) vs the simulation time in MD-simulated ES complex.
Figure 4
Figure 4
Key configurations for step 1, the nucleophilic attack by Oγ atom of Ser117. The geometries were optimized at QM/MM(B3LYP/6-31G*:AMBER) level. The key distances in the figure are in angstroms. Carbon, oxygen, nitrogen, and hydrogen atoms are colored in green, red, blue, and white, respectively. The backbone of the protein is rendered as a cartoon and colored in orange. The QM atoms are represented as ball and stick, and the surrounding residues rendered as stick. The figures below are represented using the same method.
Figure 5
Figure 5
(A) to (E) Key configurations for step 2, the dissociation of (−)-cocaine benzoyl ester. The geometries were optimized at QM/MM(B3LYP/6-31G*:AMBER) level. (F) Potential energy of step 2 is obtained at QM/MM(B3LYP/6-31G*:AMBER) level.
Figure 6
Figure 6
Key configurations for step 3, the nucleophilic attack on the benzoyl carbonyl carbon atom by a water molecule. The geometries were optimized at QM/MM(B3LYP/6-31G*:AMBER) level.
Figure 7
Figure 7
Key configurations for step 4, the dissociation of (−)-cocaine benzoyl group and Ser117 of CocE. The geometries were optimized at QM/MM(B3LYP/6-31G*:AMBER) level.
Figure 8
Figure 8
Free energy profile determined by the MP2/6-31+G*:AMBER QM/MM-FE calculations excluding the zero-point and thermal corrections for the QM subsystem. The values in parenthesis are relative free energies including zero-point and thermal corrections for the QM subsystem.
Figure 9
Figure 9
Radial distribution of counter ions (Na+) centered around Cζ atom.
Figure 10
Figure 10
Free energy barriers with (solid line) or without (dashed line) electrostatics corrections from counter ions. The energies without electrostatic corrections from counter ions are in italics while those with corrections are in bold. The zero-point and thermal corrections for the QM subsystem are included.
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