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Structure of a low-population intermediate state in the release of an enzyme product.

De Simone A, Aprile FA, Dhulesia A, Dobson CM, Vendruscolo M - Elife (2015)

Bottom Line: Enzymes can increase the rate of biomolecular reactions by several orders of magnitude.We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release.These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom.

ABSTRACT
Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product.

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Schematic illustration of the process of product release.The product (P) is released by the enzyme (E) in a process that begins in theground (or ‘locked’) state of the complex (EP), visits ametastable (or ‘unlocked’) intermediate state (EP*) andreaches the unbound state (E + P). The interactions in the ‘lockedstate’ (EP) and in the ‘unlocked state’ (EP*) areshown in light blue.DOI:http://dx.doi.org/10.7554/eLife.02777.010
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fig3: Schematic illustration of the process of product release.The product (P) is released by the enzyme (E) in a process that begins in theground (or ‘locked’) state of the complex (EP), visits ametastable (or ‘unlocked’) intermediate state (EP*) andreaches the unbound state (E + P). The interactions in the ‘lockedstate’ (EP) and in the ‘unlocked state’ (EP*) areshown in light blue.DOI:http://dx.doi.org/10.7554/eLife.02777.010

Mentions: Overall, this analysis of the structural ensembles of human lysozyme suggests that, as aconsequence of a concerted conformational transition, the enzyme explores conformationsin which the specific and tight intermolecular interactions with the substrate in itslocked state are largely lost in favour of the formation of weak and non-specificinteractions in its unlocked state. This transition is favoured by large-scaleconformational motions in which the α and β domains become closer, thussuggesting that these motions are employed by the enzyme to modulate the affinity withthe ligand. The unlocked state therefore represents an intermediate state for productrelease. In this view (Figure 3), the enzymeproduct complex (EP) populates transiently an intermediate state (EP*) that favoursthe release of the product (E + P). Thus, the analysis of the structural ensemblesthat we have determined provides evidence that large-scale conformational transitionsare employed by enzymes along their catalytic cycles including key events in the productrelease step, which often represents the rate-limiting step that governs the turnover ofthe enzyme. The difficulty for enzymes to release the products can arise from the factthat typically the latter have similar physico-chemical characteristics to thesubstrates and therefore maintain a significant affinity for the enzyme.10.7554/eLife.02777.010Figure 3.Schematic illustration of the process of product release.


Structure of a low-population intermediate state in the release of an enzyme product.

De Simone A, Aprile FA, Dhulesia A, Dobson CM, Vendruscolo M - Elife (2015)

Schematic illustration of the process of product release.The product (P) is released by the enzyme (E) in a process that begins in theground (or ‘locked’) state of the complex (EP), visits ametastable (or ‘unlocked’) intermediate state (EP*) andreaches the unbound state (E + P). The interactions in the ‘lockedstate’ (EP) and in the ‘unlocked state’ (EP*) areshown in light blue.DOI:http://dx.doi.org/10.7554/eLife.02777.010
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383205&req=5

fig3: Schematic illustration of the process of product release.The product (P) is released by the enzyme (E) in a process that begins in theground (or ‘locked’) state of the complex (EP), visits ametastable (or ‘unlocked’) intermediate state (EP*) andreaches the unbound state (E + P). The interactions in the ‘lockedstate’ (EP) and in the ‘unlocked state’ (EP*) areshown in light blue.DOI:http://dx.doi.org/10.7554/eLife.02777.010
Mentions: Overall, this analysis of the structural ensembles of human lysozyme suggests that, as aconsequence of a concerted conformational transition, the enzyme explores conformationsin which the specific and tight intermolecular interactions with the substrate in itslocked state are largely lost in favour of the formation of weak and non-specificinteractions in its unlocked state. This transition is favoured by large-scaleconformational motions in which the α and β domains become closer, thussuggesting that these motions are employed by the enzyme to modulate the affinity withthe ligand. The unlocked state therefore represents an intermediate state for productrelease. In this view (Figure 3), the enzymeproduct complex (EP) populates transiently an intermediate state (EP*) that favoursthe release of the product (E + P). Thus, the analysis of the structural ensemblesthat we have determined provides evidence that large-scale conformational transitionsare employed by enzymes along their catalytic cycles including key events in the productrelease step, which often represents the rate-limiting step that governs the turnover ofthe enzyme. The difficulty for enzymes to release the products can arise from the factthat typically the latter have similar physico-chemical characteristics to thesubstrates and therefore maintain a significant affinity for the enzyme.10.7554/eLife.02777.010Figure 3.Schematic illustration of the process of product release.

Bottom Line: Enzymes can increase the rate of biomolecular reactions by several orders of magnitude.We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release.These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom.

ABSTRACT
Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product.

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