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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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Assignments of the 1H-15N HSQC spectra of the freeand triNAG-bound states of human lysozyme.The x-axis represents the 1H (in ppm) dimension and the y-axisrepresents the 15N (in ppm) dimension.DOI:http://dx.doi.org/10.7554/eLife.02777.004
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fig1s1: Assignments of the 1H-15N HSQC spectra of the freeand triNAG-bound states of human lysozyme.The x-axis represents the 1H (in ppm) dimension and the y-axisrepresents the 15N (in ppm) dimension.DOI:http://dx.doi.org/10.7554/eLife.02777.004

Mentions: For the full assignment of human lysozyme bound to triNAG, we performed titrations of1H-15N HSQC spectra of a 200 μM sample of15N human lysozyme, which were recorded using progressive concentrationof the ligand (0, 0.3, 0.5, 0.8, 1.1, 1.6, 2.4, 3.1, 5.2, and 10 equivalents),allowing us to sample different points along the binding curve. HSQC spectra wererecorded with a spectral width of 1621 Hz and 128 points in the 15Ndimension (Figure 1—figure supplement2). Additional information was obtained using HNCA and HNCACB experimentsof a triNAG-saturated human lysozyme sample (Grzesiek and Bax, 1992; Muhandiram andKay, 1994). The HNCA experiment was carried out with the same settings asfor the free state (see above). The HNCACB experiment was carried out with a spectralwidth of 1561 Hz and 68 points in the 15N dimension and with a spectralwidth of 13,210 Hz and 72 points in the 13C dimension. These complementarydata allowed us to obtain the full assignment of the 1H-15Nspectra (Figure 1—figure supplement1).


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)

Assignments of the 1H-15N HSQC spectra of the freeand triNAG-bound states of human lysozyme.The x-axis represents the 1H (in ppm) dimension and the y-axisrepresents the 15N (in ppm) dimension.DOI:http://dx.doi.org/10.7554/eLife.02777.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: Assignments of the 1H-15N HSQC spectra of the freeand triNAG-bound states of human lysozyme.The x-axis represents the 1H (in ppm) dimension and the y-axisrepresents the 15N (in ppm) dimension.DOI:http://dx.doi.org/10.7554/eLife.02777.004
Mentions: For the full assignment of human lysozyme bound to triNAG, we performed titrations of1H-15N HSQC spectra of a 200 μM sample of15N human lysozyme, which were recorded using progressive concentrationof the ligand (0, 0.3, 0.5, 0.8, 1.1, 1.6, 2.4, 3.1, 5.2, and 10 equivalents),allowing us to sample different points along the binding curve. HSQC spectra wererecorded with a spectral width of 1621 Hz and 128 points in the 15Ndimension (Figure 1—figure supplement2). Additional information was obtained using HNCA and HNCACB experimentsof a triNAG-saturated human lysozyme sample (Grzesiek and Bax, 1992; Muhandiram andKay, 1994). The HNCA experiment was carried out with the same settings asfor the free state (see above). The HNCACB experiment was carried out with a spectralwidth of 1561 Hz and 68 points in the 15N dimension and with a spectralwidth of 13,210 Hz and 72 points in the 13C dimension. These complementarydata allowed us to obtain the full assignment of the 1H-15Nspectra (Figure 1—figure supplement1).

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