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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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Study of the N46Q/V110Q mutant.(A) Illustration of the engineered N46Q/V110Qglutamine–glutamine interactions. (B) The1H-15N-HSQC spectrum of the N46Q/V110Q mutant showsthat the mutation does not affect the structural properties of the mutant(see also Figure 4—figuresupplement 1). (C) The increase in the ability of theN46Q/V110Q mutant to release triNAG has been assessed by surface plasmonresonance (SPR) experiments.DOI:http://dx.doi.org/10.7554/eLife.02777.014
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fig4s3: Study of the N46Q/V110Q mutant.(A) Illustration of the engineered N46Q/V110Qglutamine–glutamine interactions. (B) The1H-15N-HSQC spectrum of the N46Q/V110Q mutant showsthat the mutation does not affect the structural properties of the mutant(see also Figure 4—figuresupplement 1). (C) The increase in the ability of theN46Q/V110Q mutant to release triNAG has been assessed by surface plasmonresonance (SPR) experiments.DOI:http://dx.doi.org/10.7554/eLife.02777.014

Mentions: To further validate the conclusion that the structure that we have determined of theunlocked state represents a release intermediate, we designed a second mutationalvariant to stabilise the unlocked state, rather than destabilising it as the N44Amutation. In the new mutant, N46Q/V110Q, a strong glutamine–glutamine interactionis inserted with the purpose to stabilise the ‘unlocked’ state in itsconformation (Figure 4—figure supplement3A). We have verified the folding of the mutant by NMR (Figure 4—figure supplement 3B) and measured the bindingconstants of the ligand for the unlocked state by SPR to show that it corresponds to aweaker binding affinity (Figure 4—figuresupplement 3C). While the Kd of the wild type is about 9 μM,the Kd of the N46Q/V110Q mutant is high almost beyond detection, indicatingthat the mutant essentially does not bind the substrate. These experimentally measuredbinding constants are consistent with the observation that, considering that the freeenergy of the free state is the same, the binding free energy of the locked state islarger than that of the unlocked state because the free energy of the former is lowerthan that of the latter (Figure 1).


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)

Study of the N46Q/V110Q mutant.(A) Illustration of the engineered N46Q/V110Qglutamine–glutamine interactions. (B) The1H-15N-HSQC spectrum of the N46Q/V110Q mutant showsthat the mutation does not affect the structural properties of the mutant(see also Figure 4—figuresupplement 1). (C) The increase in the ability of theN46Q/V110Q mutant to release triNAG has been assessed by surface plasmonresonance (SPR) experiments.DOI:http://dx.doi.org/10.7554/eLife.02777.014
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Related In: Results  -  Collection

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fig4s3: Study of the N46Q/V110Q mutant.(A) Illustration of the engineered N46Q/V110Qglutamine–glutamine interactions. (B) The1H-15N-HSQC spectrum of the N46Q/V110Q mutant showsthat the mutation does not affect the structural properties of the mutant(see also Figure 4—figuresupplement 1). (C) The increase in the ability of theN46Q/V110Q mutant to release triNAG has been assessed by surface plasmonresonance (SPR) experiments.DOI:http://dx.doi.org/10.7554/eLife.02777.014
Mentions: To further validate the conclusion that the structure that we have determined of theunlocked state represents a release intermediate, we designed a second mutationalvariant to stabilise the unlocked state, rather than destabilising it as the N44Amutation. In the new mutant, N46Q/V110Q, a strong glutamine–glutamine interactionis inserted with the purpose to stabilise the ‘unlocked’ state in itsconformation (Figure 4—figure supplement3A). We have verified the folding of the mutant by NMR (Figure 4—figure supplement 3B) and measured the bindingconstants of the ligand for the unlocked state by SPR to show that it corresponds to aweaker binding affinity (Figure 4—figuresupplement 3C). While the Kd of the wild type is about 9 μM,the Kd of the N46Q/V110Q mutant is high almost beyond detection, indicatingthat the mutant essentially does not bind the substrate. These experimentally measuredbinding constants are consistent with the observation that, considering that the freeenergy of the free state is the same, the binding free energy of the locked state islarger than that of the unlocked state because the free energy of the former is lowerthan that of the latter (Figure 1).

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