## 2 Results
### Study of the Conformational Stability of Gaat UK114  
To obtain information on the UK114 thermal stability, its unfolding was monitored by circular dichroism (CD) by measuring the change in ellipticity of the protein at 220 nm, which reports on the secondary structure content of the protein. An almost flat CD profile was obtained in the range of temperature from 20 to 98 \({}^{\circ}\)C, indicating that the protein conformation was not affected by the temperature increase (Figure 5). Also, other methods, such as Thermofluor analysis assays using Sypro Orange, gave similar results [45]. Since urea is known to facilitate unfolding, the CD experiments were repeated in the presence of different urea concentrations. At 2 M urea, protein unfolding started to occur, but only at high temperature (onset \(\geq\) 90 \({}^{\circ}\)C) and the transition was not completed in the monitored temperature range. Starting from 3 M urea, the unfolding curves were fully described, allowing for the determination \begin{table}
\begin{tabular}{c c c} \hline
**Amino Acid** & **K50, \(\mu\)M** & **100/K50, \(\mu\)M\({}^{-1}\)** \\ \hline Ala & 0.29 \(\pm\) 0.02 & 344 \(\pm\) 24 \\ Leu & 0.58 \(\pm\) 0.03 & 172 \(\pm\) 9 \\ L-Met & 0.41 \(\pm\) 0.03 & 208 \(\pm\) 17 \\ L-Gln & 2.56 \(\pm\) 0.11 & 39 \(\pm\) 2 \\ L-Phe & ND & 2.5 \(\pm\) 0.2 \\ L-His & ND & ND \\ \hline \end{tabular} \({}^{1}\) Data of Figure 4 were fitted to Equation (1) to obtain the indicated values of K50, i.e., the concentration of UK114 that leads to measuring an initial velocity equal to half that measured in its absence. The data obtained with L- and d-Ala and with L- and D-Leu with the corresponding amino acid oxidases were fitted together. The K50 values for the activity in the presence of the imino acid derived from L-Phe and L-His could not be determined (ND) due to their low effect, even at high UK114 concentrations. The data obtained with L-Phe were fitted to a straight line. From the calculation of the limit for [UK114]\(\rightarrow\) 0 of the first derivative of Equation (1) (100/K50), one can obtain an estimate of the percent decrease of rate of semiconvazepine formation caused by 1 nM UK114, which could be converted into an apparent turnover number by taking into account the initial velocity value measured in the absence of UK114. For D-Phe, this value corresponds to the slope obtained from the fit of the data to a straight line.  
\end{table} 
Table 1: Specificity of UK114 \({}^{1}\). Figure 5: Thermal stability of UK114. (**a**) Temperature ramps in the absence or presence of urea at the indicated concentrations; the fraction of unfolded protein was determined from the changes in ellipticity at 220 nm. (**b**) Linear relation between the T\({}_{\text{m}}\) derived from the data in panel (**a**) and urea concentrations. of melting temperatures (T\({}_{\rm m}\)). At increasing concentrations of urea, the curves gradually translated toward relatively lower temperatures, but at 7.46 M urea the T\({}_{\rm m}\) was still high (82.5 \({}^{\circ}\)C, Figure 5). Notably, the values of T\({}_{\rm m}\) measured from the thermal unfolding curves were linearly related to the urea concentration (Figure 5b). Thus, the loss of cooperative interactions seems to be directly proportional to the urea concentration in the sample. In addition, the completely unfolded protein was unable to refold, indicating that unfolding is an irreversible process for UK114 [46]. Finally, we tested the effect of perchloric acid on UK114 since this protein was first identified as a component of a perchloric acid-soluble extract of goat liver [21]. The perchloric acid-treated recombinant UK114 retained the activity and also the conformation of the untreated protein [47]. Also, freeze-drying, in the past used for the storage of the goat liver extract, did not affect the activity of the protein after reconstitution [46]. Overall, these results further highlight the extreme conformational stability of the protein.