Journal of Molecular Biology
Regular articleCrystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site1
Introduction
In healthy individuals, gluconeogenesis serves to maintain normal blood sugar levels during periods of fasting. In diabetic patients, gluconeogenesis contributes significantly to fasting hyperglycemia1, 2. The large number of individuals affected (15 million in the United States) lends urgency to the search for better treatments. Novel therapies based on reducing the flux through gluconeogenesis could help normalize fasting blood glucose levels in diabetic patients3. The rate-limiting enzyme in the gluconeogenic pathway, phosphoenolpyruvate carboxykinase (PEPCK), catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP). The high-energy phosphate bond is derived from GTP in animals, in the reversible reaction: In the reaction catalyzed by PEPCK from bacterial, fungal, and plant sources, ATP is the phosphate donor. The three-dimensional structure of the Escherichia coli enzyme has been reported, as well as its complexes with ATP and OAA analogs4, 5, 6. Although the overall sequence identity between the mammalian and bacterial enzymes is less than 20 %, a number of residues at the active site are conserved across all species, and the reaction mechanism is thought to be the same in mammals and bacteria. In order to gain insight into the specificity of the mammalian enzymes for GTP and obtain a solid basis for drug design, we have solved the crystal structures of the human enzyme alone and in complexes with a non-hydrolyzable GTP analog and with PEP (Figure 1 and Table 1).
Section snippets
Complex with GTP
The structure of PEPCK with β,γ-methylene GTP bound reveals a new binding-site for GTP, unique to the PEPCK family. The nucleotide base of the GTP analog fits snugly into a pocket on the enzyme surface (Figure 2). The aromatic residues Phe517, Phe525, and Phe530 form three walls of the pocket, with the guanine base sandwiched between the side-chains of Phe517 and Phe530. Phe517 is strictly conserved in the GTP-dependent PEPCK family; residues 525 and 530 are either Phe or Tyr in all members of
Discussion
In summary, the structures reported here provide the first glimpse of a novel GTP-binding site, unique to the GTP-dependent PEPCK family. The guanine-binding pocket is an attractive target for inhibition by small molecules, given the opportunities for forming a number of hydrogen bonds in an otherwise hydrophobic environment shielded from water. The completion of the human genome sequence has yielded a vast number of potential targets for the pharmaceutical industry, and choosing among them is
Structure determination
Recombinant human, cytosolic PEPCK was expressed as a glutathione-S-transferase (GST) fusion protein in E. coli. The GST-PEPCK fusion protein was purified by affinity chromatography using glutathione-Sepharose 4B (Pharmacia) according to the protocol supplied by Pharmacia. The GST-tag was removed by cleavage with tobacco etch virus (TEV) protease. The digestion mixture consisted of 20 mM Hepes (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.5 mg/ml of GST-PEPCK, and 167 units of TEV per mg of
Acknowledgements
We thank Chao-Min Liu and Roger Van Deroef for cell culture work. We are grateful to Maria Werner for help in characterizing the enzyme’s specificity for nucleotides.
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Edited by I. A. Wilson
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Present address: A. Swain, Biomedical Technology Area, National Center for Research Resources, 6705 Rockledge Drive, MSC 7965, Bethesda, MD 20892-7965, USA.