Projects per year
Abstract
“Heavy” (isotopically labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2H, 15N, and 13C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only “heavy enzyme” effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy–entropy compensation plays a major role in minimizing the magnitude of “heavy enzyme” isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between “light” and “heavy” enzymes on the nanosecond–picosecond time scale, suggesting relevant time scale(s) for those vibrations implicated in the “heavy enzyme” isotope effect on the PETNR reaction.
Original language | English |
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Pages (from-to) | 13693–13699 |
Journal | Journal of the American Chemical Society |
Volume | 138 |
Issue number | 41 |
DOIs | |
Publication status | Published - 27 Sept 2016 |
Research Beacons, Institutes and Platforms
- Manchester Institute of Biotechnology
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- 3 Finished
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Heavy enzymes: Probing fast dynamics in enzyme catalysis by mass modulation
1/01/15 → 31/12/17
Project: Research
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Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals
Scrutton, N., Azapagic, A., Balmer, A., Barran, P., Breitling, R., Delneri, D., Dixon, N., Faulon, J., Flitsch, S., Goble, C., Goodacre, R., Hay, S., Kell, D., Leys, D., Lloyd, J., Lockyer, N., Martin, P., Micklefield, J., Munro, A., Pedrosa Mendes, P., Randles, S., Salehi Yazdi, F., Shapira, P., Takano, E., Turner, N. & Winterburn, J.
14/11/14 → 13/05/20
Project: Research
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Linking experiment to theory: Quantum entanglement during enzyme catalysis - Dr S Hay fellowship
1/09/10 → 31/08/15
Project: Research