TEMPERATURE, pH, AND SOLVENT DEUTERIUM ISOTOPE EFFECTS; EQUILIBRIUM, STEADY STATE, AND PRE-STEADY STATE MEASUREMENTS*
2011
Temperature jump and stopped flow methods were used to study at pH 6.3 and pH 7.0 the temperature dependence of elementary steps of the reactions of lysozyme with the /3(1 + 4)-linked trimer, tetramer, and hexamer of N-acetylglucosamine. The steady state rate of cleavage of the hexasaccharide was determined as a function of temperature (5”-40”) and pH (2 to 8) in H,O solution and as a function of pD (2.5 to 9.5) at 40” in D,O solution. The apparent enthalpies of the two ionizations of apparent pK 3.8 and 6.7 observed in measurements of k,,, are 0 to 2 kcal/mol. The energy of activation determined for the pH optimum is 21.5 kcal/mol. The solvent deuterium isotope effect measured for k”,b,: at the pH (pD) optimum is 1.5 and reflects isotope effects on pre-equilibrium steps and on the rate-determining step. Transfer from H,O to D,O solution produces 0.2 to 0.4 kcal/mol more negative free energies of saccharide binding and no changes in the enthalpies of binding. Pre-steady state, steady state, and equilibrium measurements indicate a pathway for the reaction of lysozyme with hexasaccharide. The results define for this mechanism the complete free energy profile and an essentially complete enthalpy profile. Three of the five observable ES complexes are present at nearly equal concentrations. The free energies of the transition states are within a range of 3 kcal. The enthalpies of productive enzyme-substrate complexes are about 5 kcal/mol greater than the enthalpies of nonproductive complexes. Changes in tryptophan fluorescence were observed for each elementary step, and changes in pK of Glu-35 for the isomerizations of nonproductive and productive complexes. The signal changes during formation of nonproductive complexes are the same for the oligosaccharides (GlcNAc), to (GlcNAc),. The changes for productive complexes are similar but not identical with saccharides (GlcNAc), to (GlcNAc),. Correlations of the present data with previous crystallographic and solution measurements indicate the structures of productive and nonproductive
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