A transition state analog of lysozyme catalysis prepared from the bacterial cell wall tetrasaccharide.

Treatment of the cell wall tetrasaccharide GlcNAcbeta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc with alkali resulted in the formation of the unsaturated tetrasaccharide GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen. The same compound was also formed by transglycosylation upon incubation of the unmodified tetrasaccharide with the unsaturated disaccharide GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen (Tipper, D. J. (1968) Biochemistry 7, 1441-1449) and hen egg white lysozyme. The unsaturated tetrasaccharide was further characterized by paper electrophoresis, amino sugar analysis, and NMR. From NMR analysis it is concluded that the delta2,3-2-acetamido-2-deoxy-D-glucoseen at the reducing end of the unsaturated tetrasaccharide has a half-chair conformation. This conformation is similar to the one proposed for the sugar at subsite D in the lysozyme-substrate complex in the transition state. Addition of the unsaturated tetrasaccharide to a solution of hen egg white lysozyme quenched the fluorescence of the enzyme and shifted the fluorescence maximum to the blue, similar to the effect produced by the parent compound. The association constant of the unsaturated tetrasaccharide and lysozyme was measured at pH 6.0 and 24 degrees by spectrofluorimetry and microcalorimetry and found to be 1.45 X 10(5) M-1 and 2.5 X 10(5) M-1, respectively. The average value is 100 times higher than that found for the binding of unmodified tetrasaccharide to the enzyme under the same conditions. The unsaturated tetrasaccharide proved to be a better inhibitor of the lysis of Micrococcus luteus cells than the parent compound by a factor of 35. These results support the hypothesis that the active site of the enzyme is constructed so as to bind the transition state for the reaction it catalyzes more firmly than the substrate itself.