Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of "hunger hormone" ghrelin

Abstract

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

Figure 1. Possible reaction pathways for human BChE-catalyzed hydrolysis of ghrelin.

(A) Amino acid sequence of human ghrelin. (B) Acylation process (with or without existence of a tetrahedral intermediate) in which ghrelin is represented as CH₃(CH₂)₆COOR: Reactant State (RS), Tetrahedral Intermediate (TI_a), and Acyl-Enzyme (AE) + desacyl-ghrelin (ROH). (C) Deacylation process: Acyl-Enzyme (AE) + H₂O, Tetrahedral Intermediate (TI_d), and Product State (PS).

Figure 2

Optimized geometries of (A) the reactant state (RS) and (B) the transition state (TS1) for the acylation process of BChE-catalyzed hydrolysis of ghrelin. Indicated in the figures are the internuclear distances (Å) optimized at the QM/MM(SCC-DFTB:CHARMM27) level, and the values in parentheses refer to the distances optimized at the QM/MM(B3LYP/6-31G*:CHARMM27) level. (C) Plot of the potential energy vs the reaction coordinate (RC1) used the in the QM/MM(SCC-DFTB:CHARMM27) reaction-coordinate calculations; RC1 = r(C−O³) – r(C−O^γ). (D) Plots of key internuclear distances vs the intrinsic reaction coordinate (IRC) calculations at the QM/MM(SCC-DFTB:CHARMM27) level. The distances shown include the following atoms: C (carbonyl carbon atom on the n-octanoylated Ser3 side chain of ghrelin); O³ (ester oxygen atom on the n-octanoylated Ser3 side chain of ghrelin); O^γ (hydroxyl oxygen atom on Ser198 side chain of BChE); H^γ (hydroxyl hydrogen on the n-octanoylated Ser3 side chain of ghrelin); and N^ε (nitrogen atom on His438 side chain of BChE).

Figure 3

Optimized geometries of (A) the acyl-enzyme (AE) + H₂O, (B) transition state TS2, (C) tetrahedral intermediate TI_d, and (D) transition state TS3 during the deacylation process of BChE-catalyzed hydrolysis of ghrelin. Indicated in the geometries are the internuclear distances (Å) optimized at the QM/MM(SCC-DFTB:CHARMM27) level, and the corresponding values in parentheses refer to the distances optimized at the QM/MM(B3LYP/6-31G*:CHARMM27) level.

Figure 4. Minimum free energy profile determined by the QM/MM(SCC-DFTB:CHARMM27) calculations based two-dimensional PMF free energy maps for the entire reaction process (acylation and deacylation) of BChE-catalyzed hydrolysis of ghrelin.