Figure 13. Postulated catalytic cycle of EI.

Phosphoenolpyruvate (PEP) binds to the EIC domain of free EI (I, this work) leading to a conformational change that results in the formation of the phosphorylated intermediate observed crystallographically (II¹⁶). The EIN domain is in the A state conformation in structure I and the B state conformation in structure II. The HPr binding surface on the EIN^α subdomain of II is fully accessible permitting binding of HPr (structure III). The binding of HPr, together with the dissociation of the product pyruvate, induces reorientation of the EIN^α/β subdomain through a conformational change in the EIN/EIClinker (residues 255-261) resulting in the formation of a second intermediate (IV) in which the EIN domain remains in the B state conformation but the orientation of the EIN^α/β subdomain relative to EIC is the same as that found in free EI or the EI-HPr complex. Since there are very limited contacts between the EIN^α and EIN^α/β subdomains in the B state conformation, the EIN domain in structure IV rapidly relaxes to the A state through concerted conformational changes in residues 22-24 and 143-146 located in the linker regions joining the EIN^α and EIN^α/β subdomains to generate the structure of the EI-HPr complex observed experimentally (V, this work). Subsequent dissociation of HPr results in minor inward displacement of the EIN domain to form the structure of free EI (I) in which the position of the EIN domain is stabilized by a limited set of contacts with the EIC domain. In the absence of HPr, the phosphorylated intermediate (II), upon dissociation of the product pyruvate, can relax to the free EI conformation (I) via an intermediate IV′, analogous to IV but without HPr bound. The three postulated structures, III, IV and IV′, are shown in parentheses. Only a single subunit of EI is displayed. The EIC domain and the EIN^α and EIN^α/β subdomains are displayed as red, green and blue molecular surfaces, respectively; the linker helix and linker regions (connecting EIN^α to EIN^α/β, and EIN^α/β to EIC) are shown as orange ribbons; HPr is shown as a purple ribbon.