A Transition State Analog for Lysozyme

The δ-lactone derived from tetra- N -acetylchitotetraose (TACL) has been prepared by oxidation of tetra- N -acetyl-chitotetraose with iodine. The binding of TACL to lysozyme has been investigated by its inhibition of the lysozymecatalyzed lysis of Micrococcus lysodeikticus cells and by its perturbation of the tryptophyl fluorescence spectrum of lysozyme. At pH 6.2 the concentration of TACL that is required for 50% inhibition of the rate of lysis is 0.7 µ m , which is 1/110 of the concentration of the unmodified tetrasaccharide that is required for such inhibition. The association constants for the binding of TACL to lysozyme over the pH range from 2 to 8 were obtained by fluorescence measurements. Their pH dependence shows that TACL binds most strongly to the species of lysozyme in which the carboxyl group of glutamate 35 is dissociated. In agreement with this result, the fluorescence-pH profile of the TACL-lysozyme complex indicates that the pK of glutamate 35 is about 4.7 in the complex, whereas the pK of glutamate 35 in the enzyme alone is about 6.0. The value of the association constant for the binding of TACL at pH 5.0 and 25° is 3.3 x 10 6 m -1 , which is 32 times larger than that for the binding of the unmodified tetrasaccharide under the same conditions. On the basis of these results and of the similarity between the known conformation of the lactone ring and the proposed conformation of the transition state for lysozyme-catalyzed reactions (both half-chair ones), we conclude that TACL is a transition state analog for lysozyme. Furthermore, with these results we can estimate that the affinity of Subsite D of lysozyme for the half-chair conformation of the pyranose ring of N -acetylglucosamine is greater by a factor of 6 x 10 3 than its affinity for the chair conformation and thus contributes this factor to catalysis.