ArticleThe Journal of biological chemistry2012
A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB.
Article in The Journal of biological chemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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33 citing papers in PubMed, 76 citations in OpenAlex.
- Characterization of successiveApplied and environmental microbiology · 2026Article
- Molecular Access Engineering for Microbial Biocatalysis: from Enzyme Tunnels to Microbial Cell Factories.Journal of microbiology and biotechnology · 2026Review
- Elp3 uses a conserved molecular tunnel to transport acetate between distant active sites and catalyze tRNA wobble base modification.Nature communications · 2026Article
- WatCon: A Python Tool for Analysis of Conserved Water Networks Across Protein Families.JACS Au · 2025Article
- Decoding Protein Stabilization: Impact on Aggregation, Solubility, and Unfolding Mechanisms.Journal of chemical information and modeling · 2025Article
- Impact of water models on the structure and dynamics of enzyme tunnels.Computational and structural biotechnology journal · 2024Article
- Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.Angewandte Chemie (International ed. in English) · 2024Article
- Enhancing β-Galactosidase Performance for Galactooligosaccharides Preparation via Strategic Glucose Re-Tunneling.International journal of molecular sciences · 2024Article
- Reinforcing Tunnel Network Exploration in Proteins Using Gaussian Accelerated Molecular Dynamics.Journal of chemical information and modeling · 2024Article
- Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family.ACS catalysis · 2024Article
- Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.bioRxiv : the preprint server for biology · 2024Article
- Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF fromJACS Au · 2024Article
- Structural evolution of Delta lineage of SARS-CoV-2.International journal of biological macromolecules · 2023Article
- Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity.PloS one · 2023Article
- Geometry-Based versus Small-Molecule Tracking Method for Tunnel Identification: Benefits and Pitfalls.Journal of chemical information and modeling · 2022Article
- Probing the function of a ligand-modulated dynamic tunnel in bifunctional proline utilization A (PutA).Archives of biochemistry and biophysics · 2021Article
- Substrate inhibition by the blockage of product release and its control by tunnel engineering.RSC chemical biology · 2021Article
- Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.Chembiochem : a European journal of chemical biology · 2021Article
- The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step.Computational and structural biotechnology journal · 2020Article
- Identification of tunnels as in potato hydrolases.Bioinformation · 2020Article
Corrections and comments
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Authors and funding
7 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.
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