Resonance Raman spectroscopic studies of the interactions between trypsin and a competitive inhibitor.
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'-dimethylamine azobenzene, was synthesized. This compound competitively inhibits the enzyme with a 1:1 stoichiometry and an inhibition constant Ki of 2.3 muM at pH 6.08 and 15 degrees. 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.
D001391:Azo Compounds / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D007700:Kinetics; D013057:Spectrum Analysis / Q000379:methods; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*
D001391:Azo Compounds; D001555:Benzene Derivatives; D014361:Trypsin Inhibitors; D014357:Trypsin