The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tu...
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| Language: | English |
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Elsevier
2020-01-01
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| Series: | Computational and Structural Biotechnology Journal |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2001037019305598 |
| _version_ | 1818865110898180096 |
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| author | Piia Kokkonen Michaela Slanska Veronika Dockalova Gaspar P. Pinto Esther M. Sánchez-Carnerero Jiri Damborsky Petr Klán Zbynek Prokop David Bednar |
| author_facet | Piia Kokkonen Michaela Slanska Veronika Dockalova Gaspar P. Pinto Esther M. Sánchez-Carnerero Jiri Damborsky Petr Klán Zbynek Prokop David Bednar |
| author_sort | Piia Kokkonen |
| collection | DOAJ |
| description | Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4′-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair. |
| first_indexed | 2024-12-19T10:42:21Z |
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| institution | Directory Open Access Journal |
| issn | 2001-0370 |
| language | English |
| last_indexed | 2024-12-19T10:42:21Z |
| publishDate | 2020-01-01 |
| publisher | Elsevier |
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| series | Computational and Structural Biotechnology Journal |
| spelling | doaj.art-493aafb59e9347a1a3cfbd8d85f9384d2022-12-21T20:25:24ZengElsevierComputational and Structural Biotechnology Journal2001-03702020-01-0118805813The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting stepPiia Kokkonen0Michaela Slanska1Veronika Dockalova2Gaspar P. Pinto3Esther M. Sánchez-Carnerero4Jiri Damborsky5Petr Klán6Zbynek Prokop7David Bednar8Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic; International Clinical Research Centre, St. Ann’s Hospital, Brno, Czech RepublicDepartment of Chemistry and RECETOX, Faculty of Science, Masaryk University, Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic; International Clinical Research Centre, St. Ann’s Hospital, Brno, Czech RepublicDepartment of Chemistry and RECETOX, Faculty of Science, Masaryk University, Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic; International Clinical Research Centre, St. Ann’s Hospital, Brno, Czech Republic; Corresponding authors at: Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic.Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic; International Clinical Research Centre, St. Ann’s Hospital, Brno, Czech Republic; Corresponding authors at: Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic.Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4′-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.http://www.sciencedirect.com/science/article/pii/S2001037019305598Enzyme kineticsEnzyme mutationSubstrate specificity |
| spellingShingle | Piia Kokkonen Michaela Slanska Veronika Dockalova Gaspar P. Pinto Esther M. Sánchez-Carnerero Jiri Damborsky Petr Klán Zbynek Prokop David Bednar The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step Computational and Structural Biotechnology Journal Enzyme kinetics Enzyme mutation Substrate specificity |
| title | The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step |
| title_full | The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step |
| title_fullStr | The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step |
| title_full_unstemmed | The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step |
| title_short | The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step |
| title_sort | impact of tunnel mutations on enzymatic catalysis depends on the tunnel substrate complementarity and the rate limiting step |
| topic | Enzyme kinetics Enzyme mutation Substrate specificity |
| url | http://www.sciencedirect.com/science/article/pii/S2001037019305598 |
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