CRYSTAL-STRUCTURE OF ESCHERICHIA-COLI PYRUVATE-KINASE TYPE-I - MOLECULAR-BASIS OF THE ALLOSTERIC TRANSITION

Background: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate p hosphoenolpyruvate and by one or more allosteric effecters. The crysta l structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allos teric type I PK from Escherichia roll, in order to study the mechanism of allosteric regulation. Results: The 2.5 Angstrom resolution crysta l structure of the unligated type I PK in the inactive T-state shows t hat each subunit of the homotetrameric enzyme comprises a (beta/alpha) (8)-barrel domain, a flexible beta-barrel domain and a C-terminal doma in. The allosteric and active sites are located at the domain interfac es. Comparison of the T-state E. coli PK with the non-allosteric muscl e enzyme, which is thought to adopt a conformation similar to the acti ve R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degre es and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Hig hly conserved residues at the subunit interfaces couple these movement s to conformational changes in the substrate and allosteric effector b inding 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 d istortion of the phosphoenolpyruvate-binding site accounting for the l ow substrate affinity of the T-state enzyme. Conclusions: Our results suggest that allosteric control of PK is accomplished through remarkab le domain and subunit rotations. On transition from the T- to the R-st ate all 12 domains of the functional tetramer modify their relative or ientations. These concerted motions are the molecular basis of the cou pling between the active centre and the allosteric site.