Abstract
BackgroundSeveral examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.MethodsHere we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.ResultsThe NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.
Highlights
Subtilisins are serine proteases with broad substrate specificity that have found widespread use in the laundry detergent industry
There is a tendency that the d δ/dT values on the face of the protein around the active site are slightly larger than on the opposite side suggesting that the protein is most flexible around the active site (Fig. 1)
In addition to be used as a measure of the strength of hydrogen bonds, the temperature dependence of the chemical shifts may probe the existence of low populated alternative states
Summary
Subtilisins are serine proteases with broad substrate specificity that have found widespread use in the laundry detergent industry. The substrate binds in two large pockets of the groove positioning the scissile bond in the active site. Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. Our data show that there is very little structural heterogeneity in the resting state of Savinase and that Savinase does not rely on conformational selection to drive the catalytic process
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