Conformational Energy Calculations of Enzyme-Substrate and Enzyme-Inhibitor Complexes of Lysozyme. 2. Calculation of the Structures of Complexes with a Flexible Enzyme

Conformational energy calculations were carried out to investigate the most favored binding modes of oligomers of β-D-N-acetylglucosamine (GlcNAc) to the active site of lysozyme. Both the substrate and the side chains of the enzyme were allowed to undergo conformational changes and relative motions during energy minimization. It was found that, regardless of whether the side chains of the enzyme were held rigidly or were allowed to move, (GlcNAc)₆ with standard geometry had a clear preference for binding to the active site cleft with its last two residues on the “left” side of the cleft region. This region contains such residues as Arg 45, Asn 46, and Thr 47, compared with the “right” side of the cleft which contains such residues as Phe 34 and Arg 114. This result was obtained irrespective of the location of the fourth residue, i.e., irrespective of how deeply it was buried in the active site cleft. Our earlier calculation (with the enzyme held rigidly), starting with the model-built structure (from the literature), with residue 4 buried deeply in the D site on the right side of the cleft and having a half-chair conformation, led to a high-energy structure. However, when the side chains of the enzyme were allowed to undergo changes of conformation, the energy of this structure was lowered significantly because of favorable contacts on the right side of the cleft. Nevertheless, the conformational energy of this structure was still higher (by about 5 kcal/mol) than that of the most stable hexamer having standard geometry (i.e., without distortion of residue 4) and situated on the left side of the cleft. In addition, a hexamer with standard geometry could bind with the same low conformational energy as that of the energy-minimized model-built structure on the right side without distortion of residue 4. However, the conformational energy of this structure could be lowered by ∼14 kcal/mol, if distortion of the fourth residue, binding in the D site, were allowed. The conformational energy of this latter structure was the lowest of all those found in the energy search, if the strain energy for the fourth residue were ignored. If it were taken into account, however, the conformational energy of this species would probably increase so that it would become equal to or higher than that for the lowest energy hexamers binding to the left side of the active site. The finding of three low-energy structures, an undistorted mode on the left side of the active site, an undistorted mode of higher conformational energy on the right side, and a distorted mode on the right side, correlates well with recent experimental kinetic data on the binding of (GlcNAc)₆ to lysozyme. Finally, the presence of the N-acetyl group on GlcNAc provides sufficient interactions to account for the fact that GlcNAc oligomers bind to lysozyme, whereas glucose oligomers bind with much lower affinity.