| 1. |
- Amrein, Beat A., et al.
(författare)
-
Expanding the catalytic triad in epoxide hydrolases and related enzymes
- 2015
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 5:10, s. 5702-5713
-
Tidskriftsartikel (refereegranskat)abstract
- Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.
|
|
| 2. |
- Ben-David, Moshe, et al.
(författare)
-
Catalytic Stimulation by Restrained Active-Site Floppiness-The Case of High Density Lipoprotein-Bound Serum Paraoxonase-1
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:6, s. 1359-1374
-
Tidskriftsartikel (refereegranskat)abstract
- Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
|
|
| 3. |
- Carvalho, Alexandra T. P., et al.
(författare)
-
Understanding thio-effects in simple phosphoryl systems : role of solvent effects and nucleophile charge
- 2015
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 13:19, s. 5391-5398
-
Tidskriftsartikel (refereegranskat)abstract
- Recent experimental work (J. Org. Chem., 2012, 77, 5829) demonstrated pronounced differences in measured thio-effects for the hydrolysis of (thio) phosphodichloridates by water and hydroxide nucleophiles. In the present work, we have performed detailed quantum chemical calculations of these reactions, with the aim of rationalizing the molecular bases for this discrimination. The calculations highlight the interplay between nucleophile charge and transition state solvation in S(N)2(P) mechanisms as the basis of these differences, rather than a change in mechanism.
|
|
| 4. |
- Kulkarni, Yashraj S., et al.
(författare)
-
Enzyme Architecture : Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
- 2017
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 139:30, s. 10514-10525
-
Tidskriftsartikel (refereegranskat)abstract
- Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.
|
|
| 5. |
|
|
| 6. |
- Shen, Ruidan, et al.
(författare)
-
Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases
- 2022
-
Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 13:45, s. 13524-13540
-
Tidskriftsartikel (refereegranskat)abstract
- Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.
|
|
| 7. |
- Shen, Ruidan, et al.
(författare)
-
Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases
- 2021
-
Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:5, s. 646-659
-
Tidskriftsartikel (refereegranskat)abstract
- Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pKa of the nucleophilic cysteine. Compared to wild type, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations but suggest an increased preference for the WPD-loop-closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by nonchemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.
|
|
| 8. |
- Österlund, Nicklas, et al.
(författare)
-
Amyloid-beta Peptide Interactions with Amphiphilic Surfactants : Electrostatic and Hydrophobic Effects
- 2018
-
Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 9:7, s. 1680-1692
-
Tidskriftsartikel (refereegranskat)abstract
- The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.
|
|
| 9. |
- Aqvist, Johan, et al.
(författare)
-
Conserved Motifs in Different Classes of GTPases Dictate their Specific Modes of Catalysis
- 2016
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 6:3, s. 1737-1743
-
Tidskriftsartikel (refereegranskat)abstract
- The GTPase superfamily of enzymes that hydrolyze GTP have a number of conserved sequence regions (the so-called "G-motifs"), and several of the subfamilies also require catalytic activation by specific GTPase-activating proteins. In the translational GTPases involved in protein synthesis, this activating function is instead accomplished by their interaction with the ribosome. Despite these similarities, there are distinct differences regarding some of the amino acid residues making up the GTPase active sites. This raises the question of whether or not the catalytic mechanisms of different types of GTPases are identical. We report herein extensive computer simulations of both the intrinsic GTP hydrolysis reaction of Ras and the considerably faster reaction activated by the interaction with RasGAP. The results of these calculations are compared to earlier simulations of GTP hydrolysis by EF-Tu on the ribosome and show that the favored reaction pathways are strongly dependent on the composition of the active site. By computing Arrhenius plots for the temperature dependence of the calculated free energy profiles, we further show that different mechanistic pathways are associated with distinct differences in activation entropies and enthalpies. The activation parameters are in good agreement with experimental data, and we conclude that calculations of Arrhenius plots from computer simulations can be very useful for dissecting the energetics of enzyme catalysis.
|
|
| 10. |
- Ben-David, Moshe, et al.
(författare)
-
Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition
- 2020
-
Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:4, s. 1133-1147
-
Tidskriftsartikel (refereegranskat)abstract
- Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
|
|
