EVIDENCE FOR A MAJOR STRUCTURAL-CHANGE IN ESCHERICHIA-COLI CHORISMATESYNTHASE INDUCED BY FLAVIN AND SUBSTRATE-BINDING

Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyr uvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EP SP to form a stable ternary complex which does not undergo turnover. T his complex can be considered to be a model of the ternary complex bet ween enzyme, EPSP and reduced FMN immediately before catalysis commenc es. It is shown that the binding of oxidized FMN and EPSP to chorismat e synthase affects the properties and structure of the protein. Change s in small-angle X-ray scattering data, decreased susceptibility to tr yptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state . The changes in the FT-IR spectrum appear to reflect changes in the e nvironment of the secondary-structural elements rather than alteration s in their distribution, because the far-UV CD spectrum changes very l ittle. Changes in the mobility of the protein during non-denaturing PA GE indicate that the ternary complex may exhibit less conformational f lexibility than the apoprotein. Increased enzyme solubility and decrea sed tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investi gated using far-UV CD spectroscopy, and the tertiary structure was pre dicted to be an alpha-beta-barrel using discrete state-space modelling .