Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic Resonance

Indranil Duttagupta; Natacha Jugniot; Gérard Audran; Jean-Michel Franconi; Sylvain R A Marque; Philippe Massot; Philippe Mellet; Elodie Parzy; Eric Thiaudière; Nicolas Vanthuyne

doi:10.1002/chem.201800866

Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic Resonance

Chemistry. 2018 May 28;24(30):7615-7619. doi: 10.1002/chem.201800866. Epub 2018 May 3.

Authors

Affiliations

¹ Aix Marseille Univ, CNRS, ICR, UMR 7273, Case 551, Avenue Escadrille Normandie-Niemen, 13397, Marseille Cedex 20, France.
² Centre de Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS, Case 93, University Bordeaux Segalen, 146 rue Leo Saignat, 33076, Bordeaux Cedex, France.
³ N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Pr. Laurentjeva 9, Novosibirsk, 630090, Russia.
⁴ INSERM, 33076, Bordeaux Cedex, France.
⁵ Aix Marseille Univ., CNRS, ISM2, UMR 7313, Avenue Escadrille Normandie-Niemen, 13397, Marseille Cedex 20, France.

PMID: 29722459
DOI: 10.1002/chem.201800866

Abstract

A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.

Keywords: EPR spectroscopy; Michaelis constants; enzymatic activity; nitroxides; selectivity.