Structureal Analysis of Phosphoglucose Isomerase Complexes

Kathy Z Chang and Constance Jeffery, University of Illinois-Chicago, Chicago, IL, 60607

Phosphoglucose Isomerase (PGI) plays a vital role in cellular respiration: it catalyzes the isomerization of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P) in glycolysis. We are using protein x-ray crystallography to study the PGI catalytic mechanism. Crystallography, however, can only analyze static structures. A series of structures with different bound inhibitors and substrates is needed to elucidate the complete mechanism. We are presently determining two structures: a complex of PGI with G6P and a complex of PGI with the competitive inhibitor arabinose-5-phosphate (A5P).
As with most crystallography, a trial and error approach was employed to identify conditions under which PGI crystals would grow. The hanging drop vapor diffusion method was utilized to grow crystals of both complexes. A drop of a solution containing a high concentration of PGI plus the substrate or inhibitor was suspended over a reservoir containing buffer and a precipitant. Crystals formed as the conditions in the saturated solution slowly approached equilibrium.
The crystals were placed in a x-ray beam, causing scattering of the x-rays. Crystals contain a repeating unit cell that provides a sufficiently strong interference pattern for measurement by an area detector. To get a three-dimensional image, the crystal was rotated, and data were collected at each angle of rotation. Through a series of mathematical computations (i.e. Fourier Transform, Patterson, etc.), the data sets were used to calculate 3-dimensional electron density maps. We are currently refining the structure of PGI complexed with A5P, at 1.9Å resolution, by performing a series of manual adjustments alternating with computational refinements.