Abstract
Equilibrium and calorimetric studies of substrate binding to turkey egg white (TEW) lysozyme were carried out at 30degrees as a function of pH (2 to 9) and ligand size (monosaccharide to hexasaccharide of N-acetylglucosamine). Steady state kinetic measurements using the N-acetylglucosamine hexasaccharide were carried out as a function of pH (2 to 9) and temperature (20-60degrees). These experiments allow comparison of the properties of TEW lysozyme with those of the hen egg white (HEW) enzyme reported previously (Banerjee, S. K., Holler, E., Hess, G. P., and Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367, and references therein). The free energies and enthalpies of oligosaccharide binding are the same for TEW and HEW lysozymes at pH 2 but are less negative for TEW lysozyme at pH 5. The pH dependence of the binding of (GlcNAc)3 and higher oligomers to TEW lysozyme is like that for the binding of beta-methyl-N-acetylglucosaminide to TEW lysozyme. These data indicate that oligosaccharide ligands bind identically with HEW and TEW lysozymes, except for the interactions of residue 101, which is aspartic acid in the HEW protein and glycine in the TEW protein (Larue, J. N., and Speck, J. C., Jr. (1970) J. Biol. Chem. 245, 1985-1991). The pH dependence of kcat is described by apparent pK values of 3.9 and 6.8 and a maximum value of kcat of 0.135 s-1. A value of 21.0 kcal/mol was calculated for deltaH from the temperature dependence of kcat. These values and the dependence of the transglycosylation reaction on acceptor concentration are within experimental error the same as those for HEW lysozyme. The more acid pK seen in the pH rate profile reflects the ionization of Asp-52 in the lysozyme-(GlcNAc)6 complex. The pK of Asp-52 in the free protein is 0.3 pK unit lower. The essential identity of the active sites of the HEW and TEW enzymes, except for the Asp-101 interactions, allows estimation of the thermodynamic properties associated with formation of the two hydrogen bonds between Asp-101 and substrate as deltaG0 = -1.2 kcal/mol, DeltaH0 = -3.6 kcal/mol, and deltaS0 = -7.9 e.u.
Highlights
Steady state kinetic measurements using the N-acetylglucosamine hexasaccharide were carried out as a function of pH (2 to 9) and temperature (20-60’)
The equilibrium binding, calorimetric, and steady state kinetic studies reported in this paper show that HEW and turkey egg white (TEW) lysozymes possess binding and catalytic properties identical in detail except for the interactions between Asp-101 and substrate
The pH dependencies of the fluorescence emission of TEW lysozyme and of its complex with (GlcNAc)$ closely resemble those for the HEW protein and its (G~cNAc)~ complex
Summary
Equilibrium and calorimetric studies of substrate binding to turkey egg white (TEW) lysozyme were carried out at 30”. Because in the pH range 2 to 5 both Asp-101 and Asp-52 might contribute to the pH dependence of catalytic properties, it was not possible to conclude that the kinetic apparent pK reflected a single ionizing group These kinetic results and subsequent calorimetric measurements of the pH dependence of the enthalpy of binding of GlcXAc-CH, (4) can be explained most by assigning a pK value of about 3.5 to Asp-52, a more complex explanation was recognized as a possibility (3, 4). The equilibrium binding, calorimetric, and steady state kinetic studies reported in this paper show that HEW and TEW lysozymes possess binding and catalytic properties identical in detail except for the interactions between Asp-101 and substrate. The substantial identity between the binding properties of TEW and HEW lysozymes permits evaluation of the thermodynamic parameters for formation of the two hydrogen bonds between the substrate and the Asp-101 carboxylate of HEW lysozyme
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