Molecular Gating of an Engineered Enzyme Captured in Real Time

Abstract

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer–fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants (k on = 1822 s–1, k off = 60 s–1) corresponding well with those from the pre-steady-state kinetics (k –1 = 1100 s–1, k 1 = 20 s–1). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

“…This is consistent with the experimental finding that the rate-limiting step of the WT-catalyzed reaction is the release of BRE rather than Brrelease. 35 The WT enzyme also had the greatest number of BRE bound states, further supporting this conclusion.…”

“…We have previously shown that L177W undergoes slow conformational changes between open and closed states and that it is difficult for Brto exit from the closed conformation. 35 W140A/F143L/L177W/I211L displays similar conformational dynamics but over shorter time scales. When DBE is bound to the p3 tunnel or the active site, it forms favorable lipophilic interactions with the surrounding residues, reducing the likelihood that the protein will go from the closed to the open state.…”

“…The modification of the export pathways of the haloalkane dehalogenase LinB presented in our previous study showed that a single mutation blocking the main access tunnel (L177W) could induce strong SI, which was subsequently removed by modifying the auxiliary tunnel (W140A/F143L/L177W/I211L) altering the dynamics of the main tunnel (Figure 1). 35,36 To provide a deeper understanding of the mechanism of this inhibitory effect, we performed in a first step a systematic S2-4). To identify the model that fits best the kinetic observations, we analyzed all three possible scenarios, examined the error in the fitted parameters and evaluated the goodness of fit visually and computationally.…”

Substrate inhibition by the blockage of product release and its control by tunnel engineering

“…Disturbing the transport of ligands into and out of the active site by blocking the main tunnel with a bulky Trp residue (L177W) unexpectedly caused strong SI. 35,36 To investigate this effect, a new variant (LinBW140A/F143L/L177W/I211L) bearing three further mutations was generated. These three mutations opened an auxiliary access tunnel to the active site, modified the dynamics of the main tunnel, and restored the level of SI to that observed in the wild type.…”

Dynamics, a Powerful Component of Current and Future in Silico Approaches for Protein Design and Engineering

“…In ligand binding and unbinding events, especially when the binding site is deeply buried in the protein structure, ligands often have to travel tens of angstroms. Such a transport process requires a series of systematic adjustments of protein side-chains and backbones along the traversed paths that might take up hundreds of milliseconds to occur [26]. Among the slowest principal motions performed by proteins are highly organized collective translocations of whole domains, starting on microsecond timescales and with amplitudes reaching nanometers.…”