Resonance Raman spectroscopic studies of the interactions between trypsin and a competitive inhibitor

Article abstract of DOI:10.1073/pnas.72.11.4223

Raman spectroscopy was used to study the interactions between bovine trypsin and a competitive inhibitor. For this purpose, a chromophoric substrate analogue, 4 amidino 4' dimethylamino azobenzene, was synthesized. This compound competitively inhibits the enzyme with a 1:1 stoichiometry and an inhibition constant Ki of 2.3 μM at pH 6.08 and 15°. Resonance Raman spectra in aqueous solution of free or enzyme bound inhibitor were analyzed. The main spectral changes observed upon enzyme inhibitor complex formation were changes in the relative intensities of four bands (1171, 1206, 1315, 1608 cm^-1) while no large frequency shifts occurred. The binding of the inhibitor molecule to the enzyme did not induce a twisting of the phenyl groups around the N=N bond. Some modifications of the band widths are interpreted in terms of a restriction of rotational motions in the inhibitor molecule. The possible involvement of specific interactions between trypsin and the benzamidinium ion part of the inhibitor molecule is discussed.