The NMR Detection of the Distorted Intermediate in the Lysozyme Catalyzed Cleavage of Cell Wall Tetrasaccharide, Lysozyme
Patt, S.L., David Dolphin, and Sykes, B.D.
Proc. Lysozyme Conf.
Direct evidence for substrate distortion during lysozyme action was provided by NMR studies using the Fourier transform technique. The change in the dihedral angle between anomeric proton and ring proton at C2 from 60 to near 0 degrees during distortion of the sugar ring to a half-chair conformation, was reflected in an increase in the coupling const. to 3 Hz when hen egg white lysozyme (3 x 10-3 M) was mixed rapidly with the substrate N-acetylglucosaminyl-N-acetylmuramyl-N-acetylglucosaminyl-N-acetylmuramic acid (1.2 x 10-2 M). The value decreased in the course of time towards 2.5 Hz, the value for the product N-acetylglucosaminyl-N-acetylmuramic acid. The time conststant for 50% cleavage was 90 to 30 sec. Measurement of the integrated resonance of the unshifted acetyl groups and measurement of the increase in reducing power during hydrolysis gave similar time constants.