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.

Catalytic Hydrolysis Mechanism of Cocaine by Human Carboxylesterase 1: An Orthoester Intermediate Slows Down the Reaction

“…However, due to the extremely high computation workload in QM/MM molecular dynamics simulations of enzymatic reactions, low computation levels (such as semi-empirical methods, e.g., SCC-DFTB and PM6) usually have to be applied to the QM region [30]. We noticed that the major parts of computation in the two works supporting the one-step acylation/deacylation mechanism [22,23] were performed with SCC-DFTB in combination of molecular force field CHARMM27. Actually, in a work published in 2017 [21], the hCES1-catalyzed hydrolysis mechanism of d -threo-methylphenidate was studied using SCC-DFTB and the results were compared with those of DFT (B3LYP/6–31G*).…”

“…Later in the same work [21], the SCC-DFTB method was optimized by modifying the Mulliken charge and then the two-step acylation mechanism was reproduced using the modified SCC-DFTB (termed “SCC-DFTBMR”). In the computation study of hCES1-catalyzed hydrolysis of cocaine [23] and butyrylcholinesterase-catalyzed hydrolysis of ghrelin [22], the SCC-DFTB method was used without modifications. So, it might be possible that the free energy of tetrahedral intermediates of cocaine and ghrelin were also overestimated, and hence, they seemed like “tetrahedral transition states”.…”

“…However, a few recent studies have challenged the four-step catalytic hydrolysis mechanism of esterases. In 2016, a QM/MM study on the hydrolysis mechanism of ghrelin catalyzed by butyrylcholinesterase [22] found the acylation stage to be a single-step process with no tetrahedral intermediate. Again, in 2018, a two-step mechanism of hCES1-catalyzed hydrolysis of cocaine was presented [23], in which both the acylation stage and the deacylation stage are single-step processes (with no tetrahedral intermediates).…”

A Comprehensive Review of Cholinesterase Modeling and Simulation

“…Computational efforts by Zhan and coworkers showed this process to follow the two-step acylation and deacylation scheme outlined above [ 74 ]. In contrast, work by the same group to model BChE hydrolysis of ghrelin, the hunger hormone, showed a single-step acylation process [ 75 ]. Interestingly, Suarez et al modeled BChE hydrolysis of butyrylcholine, a synthetic molecule that mimics acetylcholine and for which BChE is named [ 3 ], and found that the presence of glycerol or another butyrylcholine in the active site pocket stabilizes the intermediate product after transition state 2 in the deacylation step, which is a complex of BChE with butyric acid [ 76 ].…”

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin’s biological interactions and avenues for controlling ghrelin signaling

“…Several recent studies have supported butyrylcholinesterase (BChE) as a potential ghrelin esterase within human circulation (Brimijoin et al, 2016;Chen et al, 2015Chen et al, , 2017Schopfer et al, 2015). BChE composes a large proportion of total esterase activity in human serum, and recombinantly expressed BChE can hydrolyze ghrelin to desacyl ghrelin (Schopfer et al, 2015;Yao et al, 2016). BChE knock-out mice challenged with a high fat diet exhibit larger weight gain compared to control mice, with associated implications for BChE expression involvement in body energy regulation and weight control (Chen et al, 2015;Li et al, 2008;Schopfer et al, 2015).…”