Native mass spectrometry of human carbonic anhydrase I and its inhibitor complexes
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ORIGINAL PAPER
Native mass spectrometry of human carbonic anhydrase I and its inhibitor complexes Carlotta Zoppi1 · Alessio Nocentini2 · Claudiu T. Supuran2 · Alessandro Pratesi3 · Luigi Messori1 Received: 9 June 2020 / Accepted: 30 August 2020 / Published online: 14 September 2020 © The Author(s) 2020
Abstract Native mass spectrometry is a potent technique to study and characterize biomacromolecules in their native state. Here, we have applied this method to explore the solution chemistry of human carbonic anhydrase I (hCA I) and its interactions with four different inhibitors, namely three sulfonamide inhibitors (AAZ, MZA, SLC-0111) and the dithiocarbamate derivative of morpholine (DTC). Through high-resolution ESI-Q-TOF measurements, the native state of hCA I and the binding of the above inhibitors were characterized in the molecular detail. Native mass spectrometry was also exploited to assess the direct competition in solution among the various inhibitors in relation to their affinity constants. Additional studies were conducted on the interaction of hCA I with the metallodrug auranofin, under various solution and instrumental conditions. Auranofin is a selective reagent for solvent-accessible free cysteine residues, and its reactivity was analyzed also in the presence of CA inhibitors. Overall, our investigation reveals that native mass spectrometry represents an excellent tool to characterize the solution behavior of carbonic anhydrase. Graphic abstract
Keywords Human carbonic anhydrase · Mass spectrometry · Carbonic anhydrase inhibitor · Native protein analysis Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00775-020-01818-8) contains supplementary material, which is available to authorized users. Extended author information available on the last page of the article
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Introduction Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide to bicarbonate according to the following equation: CO2 + H2O ⇆ HCO3− + H+ [1, 2]. The HCO3−/CO2 equilibrium is critical for human health, and its inhibition has been a goal of therapeutic intervention for several decades [3–6]. The majority of known CA inhibitors contain a primary sulfonamide group [7, 8]. The sulfonamide anion, R SO 2NH −, coordinates to the active-site zinc and may form hydrogen bonds with active-site amino acid residues located in the immediate vicinity further stabilizing the enzyme/inhibitor complex [9]. Owing to the importance and variety of therapeutic applications, the search for inhibitors of carbonic anhydrase is still very intensely pursued. This search is greatly assisted by a precise knowledge of the binding mode of the inhibitors to the enzyme at the atomic level. To this end, a large number of biophysical methods including NMR, X-ray crystallography, surface plasmon resonance (SPR), etc. have been exploited during the last few decades. A detailed description of these methods and of the inf
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