A NUCLEAR MAGNETIC RESONANCE STUDY OF ENZYME-INHIBITOR ASSOCIATION . USE OF PH AND TEMPERATURE EFFECTS TO PROBE BINDING ENVIROMENTS

The effects of temperature and pH on the dissociation constant of methyl 2-deoxy-2-acetamido- β-D-glucopyranoside and lysozyme have been measured. In addition the effect of temperature and pH on the chemical shifts of the acetamido and glycosidic methyl groups when bound to the enzyme have been determined. It has been shown that no conformation changes occur in the binding site of lysozyme with increases in temperature up to 53°, or as a result of pH changes in the range 2.5-10. In the absence of such changes it has been concluded that the ionizable groups which affect the binding constant and the acetamido methyl group chemical shifts are very close to the inhibitor binding site. Since it has also been demonstrated that the binding properties of lysozyme in solution, as determined by nuclear magnetic resonance spectroscopy, parallel those of the crystalline enzyme, the pKa values determined in the present study can be assigned to carboxyl residues at the binding site of the enzyme. Thus it is concluded that the pKa of glutamic acid residue 35 is 6.1 and those of aspartic acid residues 101 and 103 are 4.2 and 4.7, respectively. © 1968, American Chemical Society. All rights reserved.