| 1. |
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- Kazemi, Masoud
(författare)
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Calculations of Reaction Mechanisms and Entropic Effects in Enzyme Catalysis
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost.Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell.The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization.In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water.Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.
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| 7. |
- Ersmark, Karolina, et al.
(författare)
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C2-symmetric inhibitors of Plasmodium falciparum plasmepsin II : synthesis and theoretical predictions
- 2003
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Ingår i: Bioorganic & Medicinal Chemistry. - 0968-0896 .- 1464-3391. ; 11:17, s. 3723-3733
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Tidskriftsartikel (refereegranskat)abstract
- A series of C(2)-symmetric compounds with a mannitol-based scaffold has been investigated, both theoretically and experimentally, as Plm II inhibitors. Four different stereoisomers with either benzyloxy or allyloxy P1/P1' side chains were studied. Computational ranking of the binding affinities of the eight compounds was carried out using the linear interaction energy (LIE) method relying on a complex previously determined by crystallography. Within both series of isomers the theoretical binding energies were in agreement with the enzymatic measurements, illustrating the power of the LIE method for the prediction of ligand affinities prior to synthesis. The structural models of the enzyme-inhibitor complexes obtained from the MD simulations provided a basis for interpretation of further structure-activity relationships. Hence, the affinity of a structurally similar ligand, but with a different P2/P2' substituent was examined using the same procedure. The predicted improvement in binding constant agreed well with experimental results.
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| 8. |
- Reddy Vanga, Sudarsana, et al.
(författare)
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Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-based Inhibitors
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has been recently crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP, known as HFI series, and developed a robust computational model to explain the SAR and potentially guide the optimization of this scaffold. Our binding model positions the benzopyran ring in the catalytic binding site, coordinating the Zn+2 ion through the oxygen in position 3 of the, in contrast to previous hypothesis. The whole series of HFI compounds was systematically simulated using molecular dynamics in this binding orientation and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the demonstrated involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.
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| 9. |
- Shamsudin, Yasmin, et al.
(författare)
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Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-1
- 2015
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 54:49, s. 7283-7291
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Tidskriftsartikel (refereegranskat)abstract
- Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles.
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| 10. |
- Thomaeus, Ann, et al.
(författare)
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Active site of epoxide hydrolases revisited : A noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:9, s. 2466-2479
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Tidskriftsartikel (refereegranskat)abstract
- The carboxylate of Glu(35) in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of k(cat), and the rate of alkylenzyme hydrolysis, k(3). The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pK(a) of His(300) in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu(35) participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His(300).
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