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It was dark but the city was filled with light. Neon signs reflected off glass buildings; kaleidoscopic mirrors. Advertisements beckoned customers from multiple angles, rain-lit tarmac beamed back car headlights. Mordent was struck by the multitudes. People oozed out of subways, department stores and offices; were absorbed into subways, cars and taxis. Sharp-suited businessmen cut corners in the corners of his eyes, smart-dressed women flashed around his peripheries; everywhere Mordent looked there was movement…movement…movement. It was real life at 24 frames a second shot through a neon-noir filter and pushed into his head at a hundred miles an hour. Yet despite the skewed familiarity he craved a haven. Before leaving New York he had searched the internet to find a refuge. Their website featured an old time American jukebox with the navigational buttons animating records and leading to menus, photos, archive material and pretty girls. Mordent was a creature of habit when it came to food. Kovacs could have the job. But Kovacs was young and fresh and just on the cocky side of confident and felt he had to display all his knowledge immediately, just like Tokyo city itself. Very simple. Very fast. All the staff were Japanese. Mordent paid the taxi driver and stared. The US flag bent a rainbow over the entrance, and a cute Japanese girl in a red and white starred waistcoat and blue and white striped mini-skirt beckoned him inside. Flags jutted out from the walls every five paces in a wedding sabre arch flanked by long chrome tables that resembled ironing boards, channelling customers along the central walkway that led to the waitresses and their ever present teeth-whitened smiles. Mordent walked obediently towards the hostesses, glancing from side to side at the all-Japanese clientele holding burgers in both hands, fries piled neatly to the side, salad left untouched — more decoration than foodstuff. Yet the smell of the food teased his stomach, as though the Ghosts of Burgers Past had infiltrated his nostrils and made their way towards his intestines, an olfactory reminder of how food should really taste. She was no more than nineteen, had braces on her teeth, and a strangely shaped face that resembled playdough left in the sun. Mordent checked out his brain with the greeting and decided to satisfy himself purely from the food and the ambience. If he closed his eyes he might believe he were still in New York. No, if he closed his eyes and ears that might be the case. The Japanese language accompanied his experience as steadily as the boxset of American classic 50s hits from the jukebox. Mordent sighed as he viewed his All American Burger. He reminded himself why he was here. Why Kovacs was here. He sank into reverie as his teeth sank into the burger, satisfying his stomach if not his curiosity. They had hauled her into the precinct with a few thousand dollars worth of cocaine strapped in a package near the top of her right thigh. Mordent led the way in the search, almost finding more than she bargained for, before Kovacs burst into the interview room like a dog through a paper hoop and halted the investigation until the proper procedures could be adopted. But Mordent held back and handed the Mercado girl over for interview. It was only later, once Kovacs had gone home, that Mordent blagged his way into her holding cell for something resembling a private chat. Ruby was all lips and hair. A Latino with pasty skin, she looked like she needed to be left out longer in the sun. They had tangled before: both professionally and unprofessionally. One of the reasons why Mordent wanted to tackle her before Kovacs. You might as well blow this sky high. It was a gentle slap, a puppy slap, but a slap all the same. I can guess the ruse. The cocaine is passed through a complicated system of fake pimps, whores and johns. From A to B. Ruby smiled. Her accent fell away with her guard. Kovacs piggybacked his way to the top and I want to make sure he squeals as part of the process. Mordent stretched and looked out of the window at the Bunkyo Civic Center. From there he could see the entirety of Shinjuku, the buildings standing like students in a classroom photograph, the shorter buildings at the front and the taller ones at the back. Behind them Mount Fuji dominated the skyline with a reminder that Earth could throw up greater structures than all the architects of the world combined. Kovacs came to stand by his shoulder. Mordent pretended not to listen. The snow on the top of Mount Fuji was whiter than white. It was a clear day and not even a single cloud sullied the skyline. Rather than supplanting Kovacs, Mordent found himself bound to him. When those above contemplated sending a local team overseas so as not to appear heavy-handed with their corresponding foreign authorities the names of Kovacs and Mordent were top of the list. In that order. He knew Kovacs was quieter when he was talking. Mordent pulled out the keychain toy, laid it flat in his palm. It literally means infinite bubblewrap. You pop times and it farts. Mordent pocketed the toy. Kovacs had no idea how amused he had been to find it. But then Kovacs had no idea of his fetish. Mordent looked up from his position on the floor. The girl standing over him was naked from the waist down. Her top half was semi-see-through; her breasts distorted viewed through the overlapping layers of bubble. She squeezed her forearms against her chest and one of the skeins burst as she giggled. Mordent felt himself harden, and as she squatted over him more of the bubbles burst and he almost came there and then. He moved from pimp to whore to john: back and forth, always in that order. The network was immense and complicated. The pimp scored the cocaine and cut it. The whore needed the rush for the job. She carried it to the john who got the whore as part of the sweetener. The whore got a bonus hit from the john as a thankyou. It was a neverending case of supply and demand, with all parties being satisfied. It was always dirty, right along the line. Mr Yakamoto bowed and also shook their hands, then beckoned them to sit at the long oak table that dominated the room. Mordent noticed that Kovacs sat with his back to the view. He assumed from plain ignorance. That is, from the himo to the baishunfu to the okyakusan. This is not a new problem, of course; these types of people are always part of the connections which form a drugs ring. But from our discussions we understand that the relationship is more complex than that. There is an expression in the West, is there not, that there is nothing new under the sun? Mordent nodded. But he preferred the relationships that operated under the dusky, night-drenched glow of the moon. They left the Bunkyo Civic Center only a little wiser than when they had entered. Mr Yakamoto had advised all investigations were in hand, that the two departments — foreign to each other — would communicate through the usual channels. Kovacs had nodded in an officious, accepting manner. Mordent had also nodded, but the mugen puchipuchi popped multiple times in his pocket as they descended the elevator. Halfway down, it farted. As well as the mugen puchipuchi in his pocket he had a list of names. Kovacs eyes lit the way an educated man might raise his eyebrows at hearing a beggar speak Latin. Mercado had supplied a handful of names for contacts in Japan. It seemed that the semi-prostitutes masquerading as drug runners masquerading as prostitutes had developed their own network for staying safe. They had a Facebook group. And whilst all of them used false names and locations, Mercado was chatty enough to have made a few friends. Everyone had to have a payoff. That was how Mordent got information and Kovacs got paperwork. But whilst it lasts, the life is good. We have them to thank for our booking. Kovacs mouth hung open. The bar was tiny and unassuming. There were only seven seats in the establishment. His eyes roved around the menu. These normally have to be made months in advance. But I pulled a string. They sat at a table. Mordent watched as Kovacs accepted the situation, the beauty, delicacy and taste of the food on offer. As they ate, the temperature outside the bar dropped, the sky grew cloudy, and fat flakes hit the window and slid down the pane picking up dirt on the way, so that by the time they reached the bottom of the glass they were almost black; like cotton wool used to remove nail varnish. He passed it over to Kovacs. Watched him eat. Kovacs looked up. I got more information in one evening than he would have got all year. His tastebuds had overtaken his other senses. He was in sensory paradise. Mordent let him have his moment. There were four other occupied seats in the bar. Each contained a Japanese male of portly appearance. Gold rings adorned their fingers. The remaining seat, the one closest to Mordent and Kovacs, was unoccupied. Then the door opened and the American Mordent had watched leave the Embassy shook a light covering of snow off the shoulders of his jacket and took the remaining seat beside them. He pulled the thin plastic tube out of his pocket, was about to pull the rolled slip of paper from the inside, then thought better of it and passed it to Kovacs. Kovacs looked puzzled, but the surrealism of the situation had surpassed his usual demeanour. He placed the tube in his mouth and blew hard. Jamieson unrolled the paper and ran his eyes over the names. He raised his right hand, slowly; within seconds the four Japanese businessmen had left the sushi bar, each accompanied by their personal law enforcement officer. He nodded to the girl at the entrance, shivering in her mini-skirt. She had added thick white woollen tights to her ensemble, but would need a hug if she were going to get warm. Mordent ducked into the heated establishment and made his way down the tables to find the seat he had occupied the previous evening was empty. She placed a cool jug of Sapporo Fuyu Monogatari on the table. Mordent checked himself out a large double cheeseburger with bacon and a hefty portion of fries. Then Mordent raised his glass and muttered a word of thanks to Ruby Mercado. It was 7pm in Tokyo and so 2am in New York. Her funeral would have taken place the previous afternoon. And according to his sources it had also been snowing in Brooklyn. Find out more about author Andrew Hook here. There are no lengthy forms to fill out and we need nothing but your email. You will receive a regular newsletter but no spam. Notwithstanding the foreignness, he almost felt at home. Its dirty snow, you got that? Dirty snow. Kovacs glanced at him furiously. Kovacs nodded at the door which had swung open. The girl bit her lower lip, then sat. Kovacs grimaced. Mordent smiled, pulled it from his pocket. Our work here is done. Mordent smiled. Mordent slid a hand between her legs. We work from the inside out. The food was sour, but the bar was the sweetener. Mordent held out his hand. Kovacs opened his mouth to speak, realised it was full of sashimi, and closed it. Jamieson stood. Licked his lips. View all stories. Email Address. First Name. Last Name.

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Official websites use. Share sensitive information only on official, secure websites. Correspondence: maocaiyan mail. 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, S, H, and E, 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. 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. Keywords: human carboxylesterase 1, catalytic mechanism, QM-cluster, novel intermediate, drug metabolism. Carboxylesterases CES are a large class of enzymes responsible for hydrolysis of esters and amides, and play very important roles in the metabolism of various drugs and biosubstances \[ 1 , 2 , 3 , 4 , 5 , 6 , 7 \]. In the human body, human carboxylesterase 1 hCES1 and carboxylesterase 2 hCES2 have distinct tissue distributions and very different substrate specificities, and are known to be responsible for the metabolism of different substances or drugs \[ 3 , 6 , 8 \]. It is known that hCES1 and hCES2 have significantly different substrate specificities: hCES1 mainly prefers ester substrates with a large acyl group and a small alcohol group, whereas hCES2 favors substrates with a small acyl group and a large alcohol group \[ 6 , 8 \]. As an important esterase in the human body, hCES1 participates in the metabolism of many endogenous lipids, including cholesterol and fatty acid esters, as well as a great many commonly used drugs including cocaine, clopidogrel, oseltamivir, methylphenidate, meperidine, enalapril, etc. Because of its important roles in metabolism of lipids and drugs, hCES1 is now attracting more and more attention from researchers in multiple disciplines, including lipid metabolism, prodrug design, drug metabolism, pharmacokinetics, drug—drug interactions, etc. Since hCES1 is a crucial enzyme for the metabolism of many important lipids and drugs, it would be very valuable to clearly understand its catalytic mechanism of substrate hydrolysis reaction, to increase our understanding of properties of carboxylesterases, such as substrate-specificity and reaction rates, as well as to facilitate the development of drugs or prodrugs which need to be metabolized or activated by hCES1. For instance, Aranda et al. In , Wang et al. These results agree with the previous mechanism studies of other esterases. However, a few recent studies have challenged the four-step catalytic hydrolysis mechanism of esterases. Again, in , 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. These findings urged us to further explore the catalytic mechanism of esterases, in order to clarify the catalytic mechanisms and elucidate more details in the enzymatic reactions, which would be helpful in the study of drugs or bioactive substances metabolized by hCES1 or other esterases. For each of the two stages of the catalytic hydrolysis, i. The Gibbs free energy of each of the stationary point structures are illustrated in the free energy profile in Figure 1 , which clearly reveal that each of the two stages was a two-step process. During the acylation stage of hCES1, the catalytic residue Ser attacked the carbonyl carbon of the substrate cocaine to form an orthoester intermediate IM1, which was then cleaved to give acyl—hCES1 and a methanol molecule IM2 Figure 2. During the deacylation stage, a water molecule attacked the acyl—hCES1 complex to form another orthoester intermediate, IM3, and then the ester bond between acyl and Ser was broken to release the free enzyme and hydrolysis product Figure 3. Please see Electronic Supplementary Information for the detailed structures. Free energy profile of the whole reaction process of hCES1-catalyzed hydrolysis of cocaine. Transition states and intermediates in the acylation stage of hCES1. The ligands are shown in magenta. Non-polar residues which are far from the reaction center are not displayed. In the initial structure of hCES1-cocaine complex Reactant, Figure 2 , cocaine bound to the catalytic site and formed three hydrogen bonds to hCES1, i. These three hydrogen bonds retained in all transition states and intermediates in the acylation stage, except IM1, the orthoester intermediate. Lipophilic interactions also formed between cocaine and the adjacent non-polar residues L97, L, L, and F , which are not displayed in Figure 2 and Figure 3 for clarity. The Gibbs free energy barrier of this step was quite low 1. The carbonyl oxygen formed one hydrogen bond with GNH and another hydrogen bond with cocaine:NH to form an intramolecular six-membered ring, and therefore became more stable. Through such a proton transfer and nucleophilic addition, the negative formal charge was transferred from E in Reactant to cocaine carbonyl oxygen in IM1t. This S—H—E proton transferring chain found in this study agrees very well with the previous theories \[ 4 , 24 , 25 \]. However, interestingly, IM1t was not the final product of this step. As illustrated in the IRC of TS1 Figure 4 , the black lines , after formation of IM1t, cocaine:NH gradually migrated from the nitrogen toward the carbonyl oxygen, and finally formed an orthoester tetrahedral intermediate IM1 with no formal charges on nitrogen or oxygen atoms. The red lines are the computation results with no polarization function for hydrogens, while the black ones are the normal results. It is also worth mentioning that polarization functions of hydrogen atoms were necessary for the calculation of this proton transfer. To evaluate the effects of this omission on the DFT computation results of our system, we removed the polarization functions of hydrogen atoms involved in the proton transfer and hydrogen bonding i. This result might indicate that the polarization functions of hydrogen are important in theoretical studies of such kind of enzymatic reactions and could be useful in exploring more details of the reaction mechanism. TS2 had a relative Gibbs free energy of In TS2, cocaine:OMe was rather far from the carbonyl carbon 2. In summary, the acylation stage consisted of two steps, and the second step determined the rate of the whole acylation process with a free energy barrier of These details agree well with the previous hypotheses \[ 4 , 24 , 25 \]. The deacylation process, as illustrated in Figure 3 , was basically a reverse process of the acylation stage. In TS3, the distance between water oxygen and carbonyl carbon was 1. After crossing TS3, the water oxygen continued to approach the carbonyl carbon to form a tetrahedral intermediate, which then converted to an orthoester intermediate, IM3. The Gibbs free energy barrier of this step was The free energy barrier of this step was These details also agree well with the previous hypotheses \[ 4 , 24 , 25 \]. Step 3 determined the rate of the deacylation stage, and the free energy barrier was Step 2 determined the overall rate of the whole enzymatic hydrolysis reaction, and the activation energy E a of the whole reaction was Both the previous results \[ 23 \] and ours gave good predicted E a values of the hCES1-catalyzed hydrolysis of cocaine However, many important differences were found between these studies. The deacylation process was similar to the acylation. Such an S N 2 mechanism commonly occurred in alkyl substitutions, but was rarely found in interconversion of carboxylic acid derivatives, including transesterification and ester hydrolysis \[ 26 \]. In contrast, our studies revealed a canonical four-step mechanism, in which both the acylation and the deacylation stages consisted of two steps, the nucleophilic addition and the elimination, with two triangular pyramid transition states separated by a tetrahedral intermediate Figure 5 B. A brief comparison of previously reported cocaine hydrolysis mechanism A and our results B. The blue bonds are newly formed bonds whose length is continuously decreasing, and the red bonds are bonds to be broken whose length is continuously increasing. For simplicity, the proton transfer and the involved catalytic residues H and E are not shown here. However, we could assume that an S N 2 mechanism might be possible in the enzymatic ester hydrolysis reactions, since the catalytic residues herein, H, etc. To verify this hypothesis, we tried to search for the transition states of the S N 2 reaction process, using the model and computation method as described here, but failed. In our opinion, such different outcomes probably arose from the different models and computation methods used in these studies. 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. The proposed proton transfer between Glu and His residues was clearly observed in this study. Obviously, upon receiving a proton from S or water , H would carry a positive charge and thus become energetically less stable; so, by transferring another proton to E carboxylate anion, H would diminish its extra positive charge and reduce its energy. In the one-step acylation mechanism, H received a proton from S and immediately transferred it to the methoxy group, and the deacylation mechanism was similar to the acylation. Our study found that orthoester intermediates existed in the hCES1-catalyzed cocaine hydrolysis process, both in the acylation stage IM1 and in the deacylation stage IM3. To our knowledge, a similar mechanism has been proposed for aminoacyl migration between 1,2-diol of nucleotides \[ 34 \], but such orthoester intermediates have not been reported for enzymatic ester hydrolysis yet. The formation of orthoesters significantly reduced the energy of the intermediates, and hence elevated the free energy barriers of Steps 2 and 4 Figure 1. As illustrated in Figure 1 , if the reaction of Step 1 stopped at the tetrahedral intermediate IM1t without continuing to form orthoester intermediate IM1, the free energy barrier of Step 2 would be Even so, Step 2 would still determine the rate of the whole cocaine hydrolysis reaction. This agrees with the previous DFT computation results of ester hydrolysis mechanism catalyzed by AeCXE1 \[ 20 \], which showed that the second step cleaving of the alkoxy group from the carbonyl was the rate-determining step. Hence, without orthoester intermediates formed during the enzymatic reaction, the activation free energy E a of the whole reaction would be From a chemistry viewpoint, cleavage of the methyl ester bond of cocaine would be much easier than cleavage of the benzoate ester bond; this can also be supported by the fact that conversion of cocaine to benzoylecgonine could occur non-enzymatically in plasma, but conversion of cocaine to ecgonine methyl ester must be enzymatic \[ 38 \]. Hence, we propose that the low reaction rate of hCES1-catalyzed cocaine hydrolysis might be partially attributed to the formation of orthoester intermediate during the enzymatic reaction. Apparently, the unique chemical structure of the substrate cocaine was required during the formation of orthoester intermediates. Such an intramolecular six-membered ring was rather stable and facilitated the proton transfer from the nitrogen atom to the oxygen atom to give the orthoester intermediate IM1. It should be emphasized that, since it is difficult to determine or characterize the detailed conformation of substrate in the covalent enzyme—substrate complex, the existence of orthoester intermediates of cocaine or other similar substrates still needs to be validated in future experimental studies. Nevertheless, we expect that this hypothesis can provide insights or clues for the study of metabolism mechanisms of other hCES1 substrates, as well as the design of drugs which need to be activated or deactivated by hCES1. The intramolecular six-membered ring scaffold shown in red is chemically stable. Only the orthoester intermediate in the acylation stage is shown here for example. Furthermore, as stated above, polarization functions of hydrogen atoms were required to correctly describe the formation of orthoester intermediates. But based on this finding, we would recommend that polarization functions should be applied to hydrogen atoms, at least those directly involved in the proton transfer, to correctly describe the reaction mechanisms and to explore more details of the reaction. The X-ray crystal structure of hCES1 PDB entry ID: 1MX5 \[ 17 \] was used as the initial three-dimensional 3-D structure for the modeling, because its ligand homatropine has the same scaffold tropine to cocaine and just differs from cocaine in the sidechains. The structure of homatropine was then converted to cocaine by modifying the sidechains including adding the methoxycarbonyl group and removing the hydroxymethylene group. The nitrogen atom of cocaine was protonated and the amino acid residues were set to usual protonation states. Sidechains of a few amino acid residues were slightly adjusted and an energy-minimization was performed to relax the system. Cocaine and the surrounding amino acid residues, namely Leu97, Gly—Gly, Ser—Ala, Leu, Glu, Leu, Phe, and His, were selected to be included in the final cluster model for QM computations atoms in total. The final QM cluster model had a net charge of 0 and a multiplicity of 1. The reaction mechanism and the free energy profiles of the enzymatic reaction were calculated using conventional quantum chemistry methods. For each elementary reaction, the transition state TS was firstly obtained by geometrical optimization of the initial guess structure, and the intrinsic reaction coordinate IRC was calculated, followed by geometrical optimization to give the reactant, product, and intermediates. All the stationary point structures were then subject to vibration analysis and calculated for the thermal correction values at both K and K. The hybrid-meta GGA functional MX \[ 40 , 41 \] was used in the geometrical optimization, vibration analysis, and IRC calculation, and DFT-D3 empirical dispersion corrections zero damping \[ 42 \] were applied in the calculation. The frequency scale factor of 0. Finally, single point energy of all the stationary point structures were recalculated using the double-hybrid functional PWPB95 \[ 52 \] and basis set def2-TZVP \[ 53 , 54 \] with DFT-D3 corrections BJ damping \[ 55 \], which were added to the thermal correction values of Gibbs free energy to give the final free energies. This step of single point energy calculation was accomplished in ORCA 4. In this study, the reaction mechanism of hCES1-catalyzed hydrolysis of cocaine was investigated by the QM cluster method. Our results clearly show a four-step hydrolysis mechanism, in which both the acylation stage and the deacylation stage include two transition states and one tetrahedral orthoester intermediate. Moreover, this study found that orthoester intermediates exist in the hCES1-catalyzed hydrolysis process of cocaine, which significantly elevate the activation free energy of the reaction and decrease the reaction rate. We expect that this finding and hypothesis may provide clues or ideas for the study of hCES1-related drug metabolism and design of drugs which need to be metabolized or activated by hCES1. The structures of Reactant, transition states, intermediates, and product can be found in the Electronic Supplementary Information. Funding: M. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Find articles by Maocai Yan. Find articles by Zhen Zhang. Find articles by Zhaoming Liu. Find articles by Chunyan Zhang. Find articles by Jingchang Zhang. Find articles by Shuai Fan. Find articles by Zhaoyong Yang. Open in a new tab. 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