Publication details
Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family
| Authors | |
|---|---|
| Year of publication | 2024 |
| Type | Article in Periodical |
| Magazine / Source | ACS Catalysis |
| MU Faculty or unit | |
| Citation | |
| web | https://pubs.acs.org/doi/10.1021/acscatal.4c03503 |
| Doi | http://dx.doi.org/10.1021/acscatal.4c03503 |
| Keywords | photoswitch; azobezene; enzyme; transientkinetics; mechanism; haloalkane dehalogenase; time-resolved spectroscopy |
| Attached files | |
| Description | The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates. |
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