Structure
Volume 3, Issue 7, July 1995, Pages 729-741
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Research Article
Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition

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Abstract

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 å resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (β/α)8-barrel domain, a flexible β-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 β-barrel and C-terminal domains of each subunit, which are rotated by 17° and 15°, respectively. Moreover, the relative orientation of the four subunits differs by about 16° 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 (β/α)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme.

Conclusion 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.

Keywords

active R-state
allosteric regulation
(β/α)8-barrel
domain rotations
inactive T-state

Cited by (0)

Andrea Mattevi (corresponding author), Menico Rizzi, Martino Bolognesi and Alessandro Coda, Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.

Giovanna Valentini and M Luisa Speranza, Department of Biochemistry, University of Pavia, via Taramelli 3b, 27100 Pavia, Italy.

Martino Bolognesi, Department of Physics and IST Advanced Biotechnology Centre, University of Genova, viale Benedetto XV 10, 16132 Genova, Italy.