Limits...
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.

Show MeSH
Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).(A) Free-energy landscape as a function of the angle θ.(B) Potential energy landscape,Epot, of lysozyme in thefree state; Epot represents thecontribution of the force field used in the simulations, that is, the totalforce field without the RDC restraint term (see ‘Materials andmethods’). (C) Potential energy landscape,Epot, of the lysozyme-triNAGbound state. (D) Structure of the ‘locked state’.(E) Structure of the ‘unlocked state’.DOI:http://dx.doi.org/10.7554/eLife.02777.009
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383205&req=5

fig2: Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).(A) Free-energy landscape as a function of the angle θ.(B) Potential energy landscape,Epot, of lysozyme in thefree state; Epot represents thecontribution of the force field used in the simulations, that is, the totalforce field without the RDC restraint term (see ‘Materials andmethods’). (C) Potential energy landscape,Epot, of the lysozyme-triNAGbound state. (D) Structure of the ‘locked state’.(E) Structure of the ‘unlocked state’.DOI:http://dx.doi.org/10.7554/eLife.02777.009

Mentions: Having in mind the release of the product, we designate the global free energy minimumobserved in this study as the ‘locked state’ (i.e., release incompetent),which is centred at θ values of about 58° and Cα-RMSD values of about0.9 Å from the X-ray structure of the complex (calculated by considering secondarystructure elements only), and the other free energy minimum, which has about a 13%population under the conditions of our experiments, defined as the ‘unlockedstate’ (i.e., release competent, Figure1B). The unlocked state is a compact conformation that differs from the lockedstate by a global motion in which the α and β subunits become closer, with aθ value of about 49° in the centre of the basin. This motion generatesparticularly distorted structures with global RMSD values of about 1.5 Å from theX-ray structure. The angle θ provides a simple and effective reaction coordinateto describe the effect of triNAG binding on the energy landscape of human lysozyme(Figure 2A), which clearly illustrates how theprotein is able to explore closed conformations (i.e., θ < 50°) uponligand binding.10.7554/eLife.02777.009Figure 2.Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).


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)

Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).(A) Free-energy landscape as a function of the angle θ.(B) Potential energy landscape,Epot, of lysozyme in thefree state; Epot represents thecontribution of the force field used in the simulations, that is, the totalforce field without the RDC restraint term (see ‘Materials andmethods’). (C) Potential energy landscape,Epot, of the lysozyme-triNAGbound state. (D) Structure of the ‘locked state’.(E) Structure of the ‘unlocked state’.DOI:http://dx.doi.org/10.7554/eLife.02777.009
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).(A) Free-energy landscape as a function of the angle θ.(B) Potential energy landscape,Epot, of lysozyme in thefree state; Epot represents thecontribution of the force field used in the simulations, that is, the totalforce field without the RDC restraint term (see ‘Materials andmethods’). (C) Potential energy landscape,Epot, of the lysozyme-triNAGbound state. (D) Structure of the ‘locked state’.(E) Structure of the ‘unlocked state’.DOI:http://dx.doi.org/10.7554/eLife.02777.009
Mentions: Having in mind the release of the product, we designate the global free energy minimumobserved in this study as the ‘locked state’ (i.e., release incompetent),which is centred at θ values of about 58° and Cα-RMSD values of about0.9 Å from the X-ray structure of the complex (calculated by considering secondarystructure elements only), and the other free energy minimum, which has about a 13%population under the conditions of our experiments, defined as the ‘unlockedstate’ (i.e., release competent, Figure1B). The unlocked state is a compact conformation that differs from the lockedstate by a global motion in which the α and β subunits become closer, with aθ value of about 49° in the centre of the basin. This motion generatesparticularly distorted structures with global RMSD values of about 1.5 Å from theX-ray structure. The angle θ provides a simple and effective reaction coordinateto describe the effect of triNAG binding on the energy landscape of human lysozyme(Figure 2A), which clearly illustrates how theprotein is able to explore closed conformations (i.e., θ < 50°) uponligand binding.10.7554/eLife.02777.009Figure 2.Analysis of the interactions that stabilise the intermediate state in therelease of the product (the ‘unlocked state’).

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.

Show MeSH