Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Abstract

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl 2 , were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M -1 , respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (∆Cp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K -1 mol -1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Giant Extracellular Hemoglobin of Glossoscolex paulistus: An excellent Prototype of Biosensor and Blood Substitute

“…Binding of water to hemoglobin is the determinant step in the mechanism of allosteric regulation (Pereira et al, 2005). An analytical method known as osmotic stress has been developed based on this inclusion/exclusion process for situations of low macromolecular concentrations.…”

“…An analytical method known as osmotic stress has been developed based on this inclusion/exclusion process for situations of low macromolecular concentrations. This methodology is being extensively applied to analyze the hydration water involved in the interaction of macromolecules (Pereira et al, 2005). Furthermore, the water action upon the hemoglobin structure is deeply associated to the native hydrophobic isolation inherent to the heme pockets of hemoproteins.…”

Structural binding evidence of the trypanocidal drugs Berenil® and Pentacarinate® active principles to a serine protease model

“…It is further stabilized by contacts with the carbonyl group of Gly219, the carbonyl and hydroxyl groups of Ser190 and a buried water molecule in the base of the specificity pocket of the enzyme [1,2,5,9,13,34] (Fig. 2b-d).…”

“…Trypsin is a serine protease which is specific to the carboxyl terminus of l-lysine and l-arginine [4], being an important model for the serine proteases class [5]. Many natural peptide inhibitors of trypsin have an arginine or a lysine that binds in the specificity pocket of the enzyme, which is located in the vicinity of the catalytic triad responsible for the catalysis [6].…”

“…Additionally, isothermal titration calorimetry (ITC) assays performed on the binding of pentamidine to the enzyme allowed a complete thermodynamic description of the interaction. In the light of previously described thermodynamic data [2,5] and crystallographic structures of trypsin in complex with benzamidine-based compounds (PDB IDs: 1BJU and 1BJV [1]; 1MTS, 1MTU, 1MTV and 1MTW [19]; 1GI4, 1GI5 and 1GI6 [15]; 1QB1, 1QB6 and 1QBN [12]; 1C2F, 1C1T and 1C1Q [11]; 1PPH [20]; 1F0U [21]; 1OYQ [22]; 1K1I, 1K1J, 1K1M, 1K1N and 1K1P [13] and 1Y3U, 1Y3V and 1Y3W [23]), a comparative analysis with related complexes is presented in order to establish direct correlations between bindings mode and binding affinity in benzamidine-based platforms.…”