Binding of substrate analogues to subsites D, E, and F of hen egg-white lysozyme.
The pH dependence of the binding of dye, Beibrich Scarlet, to hen egg-white lysozyme[EC 3.2.1.17] was studied at ionic strength 0.3 and 25 degrees by following circular dichroic (CD)bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35. The effect of the binding of N-acetylglucosamine (GlcNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with dye binding. The binding constants for the stronger binding site (the upper portion of lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1 x 10(-4), 5 x 10(2), and 5M(-1) for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about --1.9, --3.3, and --2.7 kcal/mole for D, E, and F, respectively.
Binding of substrate analogues to subsites D, E, and F of hen egg-white lysozyme. The pH dependence of the binding of dye, Beibrich Scarlet, to hen egg-white lysozyme[EC 3.2.1.17] was studied at ionic strength 0.3 and 25 degrees by following circular dichroic (CD)bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35. The effect of the binding of N-acetylglucosamine (GlcNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with dye binding. The binding constants for the stronger binding site (the upper portion of lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1 x 10(-4), 5 x 10(2), and 5M(-1) for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about --1.9, --3.3, and --2.7 kcal/mole for D, E, and F, respectively.