Dataset Information

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

ABSTRACT: Human carboxylesterase 1 (hCES1) is a major carboxylesterase in the human body and plays important roles in the metabolism of a wide variety of substances, including lipids and drugs, and therefore is attracting more and more attention from areas including lipid metabolism, pharmacokinetics, drug-drug interactions, and prodrug activation. In this work, we studied the catalytic hydrolysis mechanism of hCES1 by the quantum mechanics computation method, using cocaine as a model substrate. Our results support the four-step theory of the esterase catalytic hydrolysis mechanism, in which both the acylation stage and the deacylation stage include two transition states and a tetrahedral intermediate. The roles and cooperation of the catalytic triad, S221, H468, and E354, were also analyzed in this study. Moreover, orthoester intermediates were found in hCES1-catalyzed cocaine hydrolysis reaction, which significantly elevate the free energy barrier and slow down the reaction. Based on this finding, we propose that hCES1 substrates with ?-aminocarboxylester structure might form orthoester intermediates in hCES1-catalyzed hydrolysis, and therefore prolong their in vivo half-life. Thus, this study helps to clarify the catalytic mechanism of hCES1 and elucidates important details of its catalytic process, and furthermore, provides important insights into the metabolism of hCES1 substrates and drug designing.

SUBMITTER: Yan M

PROVIDER: S-EPMC6891567 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Yan Maocai M Zhang Zhen Z Liu Zhaoming Z Zhang Chunyan C Zhang Jingchang J Fan Shuai S Yang Zhaoyong Z

Molecules (Basel, Switzerland) 20191109 22

Human carboxylesterase 1 (hCES1) is a major carboxylesterase in the human body and plays important roles in the metabolism of a wide variety of substances, including lipids and drugs, and therefore is attracting more and more attention from areas including lipid metabolism, pharmacokinetics, drug-drug interactions, and prodrug activation. In this work, we studied the catalytic hydrolysis mechanism of hCES1 by the quantum mechanics computation method, using cocaine as a model substrate. Our resul ...[more]

PMID: 31717501

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Project description:Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

Project description:Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/β-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/β-hydrolase members.

Dataset Information

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

Publications

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

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