Background: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation.
Results: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme.
Conclusions: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.