| 11. |
- Duarte, Fernanda, et al.
(författare)
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Resolving apparent conflicts between theoretical and experimental models of phosphate monoester hydrolysis.
- 2015
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:3, s. 1081-93
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Tidskriftsartikel (refereegranskat)abstract
- Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.
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| 12. |
- Duarte, Fernanda, et al.
(författare)
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The Alkaline Hydrolysis of Sulfonate Esters : Challenges in Interpreting Experimental and Theoretical Data
- 2014
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Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 79:7, s. 2816-2828
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Tidskriftsartikel (refereegranskat)abstract
- Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., Org. Biomol. Chem. 10, 2012, 8095) presented a nonlinear Bronsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its N-oxide and N-methylpyridinium derivatives. Inclusion of these compounds converts the Bronsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data.
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| 13. |
- Duarte, Fernanda, et al.
(författare)
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The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis
- 2016
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 138:33, s. 10664-10673
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Tidskriftsartikel (refereegranskat)abstract
- Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.
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| 14. |
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| 15. |
- Miton, Charlotte M., et al.
(författare)
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Evolutionary repurposing of a sulfatase : A new Michaelis complex leads to efficient transition state charge offset
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:31, s. E7293-E7302
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Tidskriftsartikel (refereegranskat)abstract
- The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono-and diester hydrolyses were only marginally affected (<= 50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E.S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (beta(leaving) (group) from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.
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| 16. |
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| 17. |
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| 18. |
- Pabis, Anna, et al.
(författare)
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Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:22, s. 3061-3081
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Tidskriftsartikel (refereegranskat)abstract
- The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer.
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| 19. |
- Pedro, Joana Reis, et al.
(författare)
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Transient gain of function of cannabinoid CB1 receptors in the control of frontocortical glucose consumption in a rat model of Type-1 diabetes
- 2020
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Ingår i: Brain Research Bulletin. - : Elsevier BV. - 0361-9230. ; 161, s. 106-115
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Tidskriftsartikel (refereegranskat)abstract
- Here we aimed to unify some previous controversial reports on changes in both cannabinoid CB1 receptor (CB1R) expression and glucose metabolism in the forebrain of rodent models of diabetes. We determined how glucose metabolism and its modulation by CB1R ligands evolve in the frontal cortex of young adult male Wistar rats, in the first 8 weeks of streptozotocin-induced type-1 diabetes (T1D). We report that frontocortical CB1R protein density was biphasically altered in the first month of T1D, which was accompanied with a reduction of resting glucose uptake ex vivo in acute frontocortical slices that was normalized after eight weeks in T1D. This early reduction of glucose uptake in slices was also restored by ex vivo treatment with both the non-selective CB1R agonists, WIN55212−2 (500 nM) and the CB1R-selective agonist, ACEA (3 μM) while it was exacerbated by the CB1R-selective antagonist, O-2050 (500 nM). These results suggest a gain-of-function for the cerebrocortical CB1Rs in the control of glucose uptake in diabetes. Although insulin and IGF-1 receptor protein densities remained unaffected, phosphorylated GSKα and GSKβ levels showed different profiles 2 and 8 weeks after T1D induction in the frontal cortex. Altogether, the biphasic response in frontocortical CB1R density within a month after T1D induction resolves previous controversial reports on forebrain CB1R levels in T1D rodent models. Furthermore, this study also hints that cannabinoids may be useful to alleviate impaired glucoregulation in the diabetic cortex.
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| 20. |
- Repic, Matej, et al.
(författare)
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Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine
- 2014
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 82:12, s. 3347-3355
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Tidskriftsartikel (refereegranskat)abstract
- Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol(-1) compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol(-1), in excellent agreement with the experimental value of 16.5 kcal mol(-1). Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction.
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