2025-03-25 筑波大学
<関連情報>
- https://www.tsukuba.ac.jp/journal/medicine-health/20250325140000.html
- https://www.tsukuba.ac.jp/journal/pdf/p20250325140000.pdf
- https://www.pnas.org/doi/10.1073/pnas.2418926122
ラベルフリーin-situ電気化学による細胞内呼吸酵素動態の解明 Decoding in-cell respiratory enzyme dynamics by label-free in situ electrochemistry
Yoshihide Tokunou, Tomohiko Yamazaki, Takashi Fujikawa, and Akihiro Okamoto
Proceedings of the National Academy of Sciences Published:March 21, 2025
DOI:https://doi.org/10.1073/pnas.2418926122
Significance
Understanding enzyme function in living organisms is vital for advancing biochemistry and therapeutic strategies. However, traditional in vivo enzyme assays have primarily focused on enzymes unrelated to complex respiration. This study introduces a pioneering approach to measure Michaelis–Menten kinetic parameters of respiratory enzymes within cells. By simply manipulating rate-limiting steps in microbial current generation via direct or indirect electron transfer mechanism, our real-time electrochemical assay revealed in vivo enzyme kinetics. Utilizing gene-knockout strains and inhibitors, we demonstrated that interprotein interactions shape enzyme activity, challenging the longstanding belief that molecular crowding accounts for differences between in vivo and in vitro kinetics. This breakthrough deepens our understanding of enzyme biochemistry and potentially transforms approaches in biomedical science.
Abstract
Deciphering metabolic enzyme catalysis in living cells remains a formidable challenge due to the limitations of in vivo assays, which focus on enzymes isolated from respiration. This study introduces an innovative whole-cell electrochemical assay to reveal the Michaelis–Menten landscape of respiratory enzymes amid complex molecular interactions. We controlled the microbial current generation’s rate-limiting step, extracting in vivo kinetic parameters (Km, Ki, and kcat) for the periplasmic nitrite (NrfA) and fumarate (FccA) reductases. Notably, while NrfA kinetics mirrored those of its purified form, FccA exhibited unique kinetic behavior. Further exploration using a mutant strain lacking CymA, a periplasmic hub protein, revealed its crucial role in modulating FccA’s kinetics, challenging the prevailing view that molecular crowding is the main cause of discrepancies between in vivo and in vitro enzyme kinetics. This platform offers a groundbreaking approach to studying cellular respiratory enzymatic kinetics, paving the way for future research in bioenergetics and medicine.
