Paired Spectroscopic and Crystallographic Studies of Proteases

Abstract

The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X‐ray crystallographic studies. The active site serines of the proteases were reacted with 4‐cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water‐soluble inhibitor model, potassium 4‐cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H2O/DMSO and the frequency‐temperature line‐slope (FTLS) method in H2O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin‐CBS and trypsin‐CBS in aqueous buffer were both measured to be −3.5x10−2 cm−1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active‐site nitriles in both proteases are mostly solvated. The X‐ray crystal structures of the subtilisin‐CBS and trypsin‐CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin‐CBS and in one conformation in the trypsin‐CBS. The crystallographic data support the FTLS data that the active‐site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X‐ray crystallography provides a powerful approach to studying protein structure.

Purified proteases of two Antarctic bacteria: from screening to characterization

“…Its control, subtilisin, was inhibited by PMSF but not by EDTA, indicating subtilisin as being a serine protease. Other studies showed the presence of calcium ions in the subtilisin structure, as determined by X-ray crystallography (Luo et al 2019). Calcium ions are also described as structural stabilizers in some enzymes, such as subtilisin and thermolysin.…”