The effect of tri-N-acetylglucosamine on hydrogen exchange in hen egg white lysozyme.

Tritium-hydrogen isotope exchange techniques have been employed to study the effect of tri-N-acetylglucosamine binding on the conformational dynamics of hen egg white lysozyme. Numerical Laplace inversion of the data provides exchange rate probability density functions that reveal three overlapping peaks for both the free enzyme and (GlcNAc)3-enzyme complex. Binding of (GlcNAc)3 decreases the exchange rates of all protons to some extent with by far the largest effect being observed for the slow exchanging protons. These have been located, by comparison with neutron diffraction results (Mason, S. A., Bentley, G. A., and McIntyre, G. J. (1984) in Neutrons in Biology (Schoenborn, B. P., ed) pp. 323-334, Plenum Press, New York), within the beta-sheet structure and on helices (8-13), (28-34), and (89-97), that define the edges of the so-called "hydrophobic box" in lysozyme. The regions of the protein that are most affected by binding (GlcNAc)3, as revealed by hydrogen exchange, are found to be quite distinct from the regions observed to undergo conformational changes by x-ray diffraction. Most of these segments of the protein are located at some distance from the (GlcNAc)3-binding site itself. Two segments (the beta-sheet and helix (28-34)) are closely associated with the two active-site carboxylate groups. These results suggest that exchange-stable regions having strong, highly organized hydrogen bonding may have an important role in catalytic function and the differential propagation of conformational and dynamic perturbations caused by ligand binding at distant sites on the protein.