To characterize the hydrogen-bonding network in lysozyme, we focused on the residue of Asp48 located at the active site in hen egg-white lysozyme. We constructed a mutant lysozyme (D48A) and analyzed using (GlcNAc)3 and chitin-affinity chromatography. The substrate binding of subsites D-F in D48A and the activity against (GlcNAc)5 were decreased. The parameters of substrate binding and rate constants obtained from computer simulations confirmed these changes. In the crystal structure, (GlcNAc)4 was located at the same position as wildtype. However, the side chains of Arg45 and Thr47 at subsites E-F were moved by the replacement. Further, the loss of the hydrogen bond between Asp48 and Ser50 changed the hydrogen-bonding network, and this resulted in an alteration of the side chain of Asn59. This result suggests that the hydrogen-bonding network plays a crucial in the function of Asp52 and of transglycosylation at subsites E-F.
Keywords: crystallization; enzymatic activity; hydrogen-bonding network; lysozyme; substrate binding.