Structure of a low-population intermediate state in the release of an enzyme product
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 stabilize 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 the product.
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© 2015, De Simone et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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BTB (bric-a-brack, Tramtrack, and broad complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non-TTK-type BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through a dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.
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