Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation.
The thermal and guanidine hydrochloride (GdnHCl) denaturation of lysozyme has been investigated at various concentrations of tri-N-acetylglucosamine ((GlcNAc)3), a trisaccharide which binds specifically at the active site of native lysozyme. The presence of (GlcNAc)3 leads to a readily observable stabilization of the protein to thermal and GdnHCl denaturation. An analysis of guanidine hydrochloride denaturation curves shows that the stability of lysozyme is increased by 495 cal/mol by the presence of 3 x 10(-4) M (GlcNAc)3. The midpoint of the thermal denaturation curve, T 1/2, is increased 1.6 and 5.3 degrees C by 2.02 x 10(-4) M and 1.38 x 10(-3) M (GlcNAc)3, respectively. This corresponds to an increase in the stability of lysozyme of 385 and 1275 cal/mol. These results are in excellent agreement with predictions based on an equation derived by Schellman ((1975) Biopolymers 14, 999-1018) to take into account the effect of ligand binding on the melting temperature of a protein. delta T 1/2 = TT0R divided by delta HD ln (1 + KB[S]) where T and T0 are T1/2 values in the presence and absence of (GlcNAc)3, delta HD is the enthalpy of denaturation in the presence of (GlcNAc)3, KB in the equilibrium constant for the binding of (GlcNAc)3 to lysozyme, and [S] is the free concentration of (GlcNAc)3. Thus, the increased stability of an enzyme in the presence of its substrate, coenzyme, or any small molecule that it binds specifically results because binding to the native state shifts the unfolding equilibrium and decreases the concentration of unfolded states of the enzyme. It is suggested that this may be a more important factor than substrate-induced conformational changes in acccounting for the decreased rates of protein catabolism frequently observed in vivo at elevated substrate concentrations.
D000117:Acetylglucosamine; D001665:Binding Sites; D004355:Drug Stability; D006146:Guanidines; D007700:Kinetics; D009113:Muramidase; D009844:Oligosaccharides; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D009844:Oligosaccharides; D009113:Muramidase; D000117:Acetylglucosamine