| 1. |
- Bjelic, Sinisa, et al.
(författare)
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Cold adaptation of enzyme reaction rates
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:38, s. 10049-10057
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Tidskriftsartikel (refereegranskat)abstract
- A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.
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| 2. |
- Bjelic, Sinisa, et al.
(författare)
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Computational inhibitor design against malaria plasmepsins
- 2007
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 64:17, s. 2285-2305
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Tidskriftsartikel (refereegranskat)abstract
- Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.
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| 3. |
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| 4. |
- Johansson, Denny, 1980, et al.
(författare)
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Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:27, s. 9475-7
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Tidskriftsartikel (refereegranskat)abstract
- Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --> O or N --> S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.
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| 5. |
- Kazemi, Masoud, et al.
(författare)
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Mechanistic alternatives for peptide bond formation on the ribosome
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:11, s. 5345-5354
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Tidskriftsartikel (refereegranskat)abstract
- The peptidyl transfer reaction on the large ribosomal subunit depends on the protonation state of the amine nucleophile and exhibits a large kinetic solvent isotope effect (KSIE similar to 8). In contrast, the related peptidyl-tRNA hydrolysis reaction involved in termination shows a KSIE of similar to 4 and a pH-rate profile indicative of base catalysis. It is, however, unclear why these reactions should proceed with different mechanisms, as the experimental data suggests. One explanation is that two competing mechanisms may be operational in the peptidyl transferase center (PTC). Herein, we explored this possibility by re-examining the previously proposed proton shuttle mechanism and testing the feasibility of general base catalysis also for peptide bond formation. We employed a large cluster model of the active site and different reaction mechanisms were evaluated by density functional theory calculations. In these calculations, the proton shuttle and general base mechanisms both yield activation energies comparable to the experimental values. However, only the proton shuttle mechanism is found to be consistent with the experimentally observed pH-rate profile and the KSIE. This suggests that the PTC promotes the proton shuttle mechanism for peptide bond formation, while prohibiting general base catalysis, although the detailed mechanism by which general base catalysis is excluded remains unclear.
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| 6. |
- Andér, Martin, 1979-
(författare)
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Computational Analysis of Molecular Recognition Involving the Ribosome and a Voltage Gated K+ Channel
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel. The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center. Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified. The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex. A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted. Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.
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| 7. |
- Boukharta, Lars, 1979-
(författare)
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Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target. In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.
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| 8. |
- Guo, Xiaohu, et al.
(författare)
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Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.
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| 9. |
- Sočan, Jaka
(författare)
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Enzyme Cold Adaptation through Evolution of Protein Flexibility
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- What lives, evolves. Macromolecular catalysis is a process, central to both evolutionary and metabolic aspect of life, as it provides a systematic bias favouring certain chemical processes. In living organisms, this bias is crucial for maintenance of constant composition outside of equilibrium with the environment, and transfer of hereditary information. Biochemical catalysis is based on facilitating the formation of the least probable state along the reaction pathway and is achieved by providing a suitable environment for that. High conformational versatility of their protein scaffold is the main reason why the enzymes perform a predominant part of cellular catalytic activity.Cold ecosystems pose a hard evolutionary challenge to the organisms populating them due to significant enzymatic rate retardation with decreasing temperature. At the same time, selective pressure for protein thermal stability is generally relieved in those organisms. Compared to their mesophilic and thermophilic (warm- and heat-active) counterparts, psychrophilic (cold-active) enzymes typically show a trade-off between activity and stability, with lower melting temperatures, but more favourably distributed activation parameters. As a decreased activation enthalpy and a more negative activation entropy enable a lower free energy penalty at low temperatures, the cold-active enzymes lose significantly less catalytically activity there. Such redistribution of thermodynamic parameters is generally attributed to the favourable dynamic patterns associated with a higher flexibility of the less ordered protein regions.In the present thesis, two different strategies of attaining enzyme psychrophilicity were explored. In our comparison of aliphatic tripeptide substrates breakdown by cold adapted salmon pancreatic elastase and its mesophilic ortholog, a significant shift between activation entropy and enthalpy of around 10 kcal/mol was observed. Notably, the structure of the psychrophilic elastase was also found to be more flexible at the protein surface. Our calculations have shown that the mutants of psychrophilic elastase, including certain mesophilic structural features in the surface loops, adopt significantly less cold-active character. The effect of these features, however, does not to appear to be simply cumulative.Our investigation of the orthologous pair of starch-degrading α-amylases revealed the psychrophilic ortholog originating from Antarctic bacteria only partly relies on redistribution of activation parameters to achieve favourable rates at low temperature. Curiously, this enzyme’s activity was found to be deteriorating at the temperatures significantly lower than the enzyme’s melting temperature. The computational reproduction of the temperature optimum for cold-active α-amylase enabled us to relate this phenomenon to a deterioration of prominent substrate stabilizing interaction with increasing temperatures of the environment. We provided a simple kinetic model incorporating two reactant states in equilibrium, where only the properly stabilized reactant state is catalytically active.
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| 10. |
- Wallin, Göran, et al.
(författare)
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Folding-Reaction Coupling in a Self-Cleaving Protein
- 2012
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 8:10, s. 3871-3879
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Tidskriftsartikel (refereegranskat)abstract
- Backbone torsional strain has been implicated as a cause of rate enhancement in a class of autoprocessing proteins performing proteolysis and protein splicing. In the autoproteolytic protein domain SEA, folding and proteolytic activity have experimentally been shown to be coupled with about 7 kcal/mol of folding free energy available for catalysis. Here, we have examined the catalytic strategy of SEA with molecular dynamics simulations, potential of mean force free energy profiles, and B3LYP/6-311G(d,p) density functional calculations. A quantitative estimate of the free energy stored as protein strain (about 8 kcal/mol), that is available for catalyzing the cleavage reaction, is obtained and found to be in excellent agreement with thermodynamic and kinetic data. It is further shown that there is strong coupling between folding and reaction coordinates leading to reactant state destabilization in the direction of folding and transition state stabilization along the reaction coordinate. This situation is different from the preorganized active site model in that the fully folded transition state stabilizing structure is not realized until the reaction barrier is surmounted.
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