Crystal Structure of the Rat Liver Fructose-2,6-bisphosphatase Based on Selenomethionine Multiwavelength Anomalous Dispersion Phases^,

Abstract

The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution. The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein. Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium. The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta-strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix. The active site pocket is funnel-shaped. The narrow opening of the funnel is wide enough for a water molecule to pass. The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle. The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141. The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase. However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase. The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain. In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family.

Reaction Mechanism of Fructose-2,6-bisphosphatase

“…The crystallographic data of the rat testis enzyme (2), as well as a truncated form of the liver enzyme containing only the Fru-2,6-Pase domain (7) 307 , and His 392 , respectively, of the liver isozyme) are also located in the active site of the Fru-2,6-Pase (Fig. 1).…”

Tissue-specific Structure/Function Differentiation of the Liver Isoform of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

“…The determination of crystal structures of the rat liver Fru-2,6-P 2 ase domain (6) and the rat testis 6-Fru(P)-2-kinase/Fru-2,6-P 2 ase (7) has led a great progress in our understanding of the catalytic mechanisms of this enzyme system (8,9). However, one of the most fundamental questions about this enzyme system has remained unanswered.…”

Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain

“…This tetrapeptide sequence probably interacts directly with residues in the catalytic core of Fru-2,6-P # ase and reduces the rate of breakdown of the phosphoenzyme intermediate or the rate of product release. Based on the crystal structures of the rat testis bifunctional enzyme and the rat liver bisphosphatase domain [12,24], we postulated previously that the spatial proximity of the C-terminal tail and loop 2 of the Fru-2,6-P # ase catalytic core, which contains 11 acidic amino acids, might underlie the inhibitory effect of the C-terminal tail on the Fru-2,6-P # ase activity [15]. The results of the present work further support our idea, by suggesting that electrostatic interactions between the three basic C-terminal residues (His%%%, Arg%%& and Arg%%&) and the acidic residues within the loop 2 underlie the repression of Fru-2,6-P # ase activity by the C-terminal tail.…”