Erosantonio Lampitella, Nicola Landi, Rosario Oliva, Sara Ragucci, Luigi Petraccone, Rita Berisio, Antimo Di Maro, Pompea Del Vecchio, Lampitella, E., Landi, N., Oliva, R., Ragucci, S., Petraccone, L., Berisio, R., Di Maro, A., Del Vecchio, P., Lampitella, Erosantonio, Landi, Nicola, Oliva, Rosario, Ragucci, Sara, Petraccone, Luigi, Berisio, Rita, Di Maro, Antimo, and Del Vecchio, Pompea
Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+). Here we investigated the conformational stability of ageritin in the pH range 4.0-7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol-1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn2+. We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol-1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability.