W0199

Structure of Pyruvate Kinase with a Novel Allosteric Activator Identified via Computational Docking. Sasi K. Chilukuri, Bernard D. Santarsiero, Andrew D. Mesecar, Center for Pharmaceutical Biotechnology, Dept. of Medicinal Chemistry and Pharmacognosy, Univ. of Illinois at Chicago, IL 60607, USA.

The tissue-specific expression and regulation of pyruvate kinase (PK) is involved in at least two different human diseases. Tumor-specific pyruvate kinase (Type-M2) is overexpressed in a variety of human cancers including brain, kidney and breast. Hereditary point mutations in human erythrocyte pyruvate kinase (Type-R) lead to pyruvate kinase deficiency resulting in non-spherocytic hemolytic anemia. Our lab is currently attempting to discover novel allosteric effectors of PK that could potentially serve as lead compounds for eventual therapeutic compounds to treat these diseases. We recently discovered a series of novel allosteric activators of yeast and human R-type PK, including ribulose 1,5-bisphosphate (RuBP), via computational docking studies coupled with high throughput kinetic assays. To understand the molecular basis for allosteric activation of pyruvate kinase by RuBP, we have crystallized the yeast enzyme in complex with RuBP, phosphoglycolate, Mn²⁺ and K⁺ at pH 7.5. X-ray data were collected to 2.9 Aº resolution and to 95.7% completeness with a final R_merge of 12% and average I/σI of 11.6. The enzyme complex crystallized in space group P4₂2₁2 which is different than our previously reported space group for yeast PK co-crystallized with fructose 1,6-biphosphate (FBP), the physiological effector, and phosphoglycolate, Mn²⁺ and K⁺ at pH 6.5. We were able to crystallize the yeast PK-FBP- phosphoglycolate, Mn²⁺ and K⁺ complex in space group P4₂2₁2 by adjusting the pH to 7.5, and we collected a data set on this crystal to 2.9 Aº. The unit cell dimensions for both crystals were approximately (a=b=98, c=232). Both structures are currently in the processes of being refined, and despite crystallizing in the same space group with similar unit cell dimensions, we observe large conformational differences between the two complexes in electron density maps. The implications of these structural differences in reference to the different physical-chemical properties of these allosteric efforts will be discussed. This research is funded by a grant from the American Heart Association (AHA# 0235416Z).