Thermodynamic Analysis for Binding of 4- O-β-tri- N-acetylchitotriosyl Moranoline, a Transition State Analogue Inhibitor for Hen Egg White Lysozyme

Abstract

4-O-β-tri-N-acetylchitotriosyl moranoline (GN₃M) is a transition-state analogue for hen egg white lysozyme (HEWL) and identified as the most potent inhibitor till date. Isothermal titration calorimetry experiments provided the thermodynamic parameters for binding of GN₃M to HEWL and revealed that the binding is driven by a favorable enthalpy change (ΔH° = -11.0 kcal/mol) with an entropic penalty (-TΔS° = 2.6 kcal/mol), resulting in a free energy change (ΔG°) of -8.4 kcal/mol [Ogata et al. (2013) 288, 6,072-6,082]. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔS _solv° = -9.2 kcal/mol) is its sole contributor. The change in heat capacity (ΔC _p°) for the binding of GN₃M was determined to be -120.2 cal/K·mol. These results indicate that the bound water molecules play a crucial role in the tight interaction between GN₃M and HEWL.

Keywords: 4-O-β-tri-N-acetylchitotriosyl moranoline; binding; inhibitor; lysozyme (HEWL); thermodynamics.

FIGURE 1

Chemical structure of GN₃M and (GlcNAc)₃.

FIGURE 2

ITC thermogram (upper) and theoretical fit to the experimental data (lower) for binding of GN₃M at 20°C. Inset shows the bar diagram of the thermodynamic parameters (A). Temperature dependence of GN₃M binding to HEWL; the plot of ΔH° versus temperature yielded a change in the heat capacity (ΔC _p°) based on the slope of the line. The ΔC _p° value was calculated to be −120.2 cal/K·mol (B).

FIGURE 3

Crystal structures of (GlcNAc)₃-liganded and GN₃M-liganded HEWL (A) Stereo view of superimposed structures of (GlcNAc)₃-liganded HEWL (green; PDB code 1lzb) and GN₃M-liganded HEWL (cyan; PDB code 4hp0) complexes. The catalytic residues Glu35 and Asp52 of HEWL are indicated as sticks (GlcNAc)₃ and GN₃M are shown as orange and yellow sticks, respectively. (B) The binding modes of (GlcNAc)₃ and GN₃M to HEWL. Amino acid residues involved in the binding of ligands (GlcNAc)₃ and GN₃M are also indicated as sticks. The numbers, −4 to −1, indicate the subsite positions. Dashed lines indicate the possible hydrogen bonds. Red spheres represent oxygen atoms of water molecules.