Visualization of Enzymatic Reaction by Time-resolved Structural Analysis with Photosensitive Caged Substrate

Yakugaku Zasshi. 2022;142(5):487-494. doi: 10.1248/yakushi.21-00203-2.ABSTRACTElucidation of molecular mechanism for metalloeznyme-catalyzed reactions is longstanding topics in the bio-inorganic chemistry field, since metalloeznymes play pivotal roles in various biological processes and catalyze reactions with high efficiency under mild conditions. Structural determination of reaction intermediates is key to visualizing the reactions catalyzed by metalloenzymes. Although it is not easy to determine the structure of the transient species by conventional crystallography, newly developed time-resolved X-ray crystallography using X-ray free electron laser (XFEL) has a great potential for the structural characterization of intermediates. However, XFEL-based time-resolved crystallography requires a photo-trigger, hampering its application to non-photosensitive proteins like metalloenzymes. Here, to overcome this issue, we focused on caged substrates, which produce the substrate upon photo-irradiation, to introduce the photo-trigger into the reaction catalyzed by the metalloenzyme. To demonstrate the usefulness of caged substrates for the trigger of XFEL-based time-resolved crystallography, soluble nitric oxide (NO) reductase, which catalyzes the reduction of NO to nitrous oxide (N2O) at a heme active center, and caged NO were used as a model system. Time-resolved spectroscopic analysis showed that the photolysis of caged NO could initiate NO reduction by P450nor in the micro-crysta...
Source: Yakugaku Zasshi : Journal of the Pharmaceutical Society of Japan - Category: Drugs & Pharmacology Authors: Source Type: research