Molecular basis of the fructose‐2,6‐bisphosphatase reaction of PFKFB3: Transition state and the C‐terminal function

Abstract

The molecular basis of Fructose-2,6-bisphosphatase (F-2,6-P2ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF4), and in a complex with pyrophosphate (PFKFB3•PPi) at resolutions of 2.45Å, 2.2Å, and 2.3Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF4 and PFKFB3•PPi complexes were obtained by soaking. The PFKFB3•AlF4 and PFKFB3•PPi complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ~5.1Å, and the pivotal point 2-P is on the same line, suggesting an “in-line” transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygens of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440–446) was rearranged in PFKFB3•PPi, implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P2ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3)

“…There are four PFKFB isoforms (PFKFB1-4), of which PFKFB3 exhibits the highest kinase:phosphatase activity of all (~700-fold). PFKFB3 is able to produce F26BP at a high rate and thus drives the Warburg effect [23,24]. Under hypoxic conditions, this kinase:phosphatase activity can be increased up to 3000-fold following phosphorylation at Ser460 by PKA or AMPK [25].…”

IKKβ promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3

“…Although the observed increase in PFKFB3 activity of S269A mutant could be explained by protein level, the expression of wild-type and S269D mutant PFKFB3 is similar, yet the activity of the S269D mutant is 80% reduced. Ser269 is located in a highly disordered region critical for PFKFB3 enzyme function, as the nearby residues must remain in contact with two-phosphate oxygens and help stabilize a tele-phosphohistidine intermediate (Cavalier et al 2012;Boyd et al 2015); therefore, phosphorylation could disrupt these interactions and normal function, which could ultimately decrease the activity. Taken together, our data suggest a dual consequence of IKKβ-dependent phosphorylation of PFKFB3 Ser269: (1) acutely reducing its activity and (2) marking it for ubiquitin-mediated degradation.…”

PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2)

“…The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms. This residue is said to interact with the 2-phosphate and further stabilizes the transitions state (Cavalier et al, 2012;Kim et al, 2006).…”

“…These isoforms share highly conserved core catalytic domains (85%) but differ greatly in their kinetic properties and responses to regulatory signals (Okar et al, 2001). These differences are mostly due to highly divergent Nand C-terminal regulatory domains; however, a few but significant sequence differences in the catalytic domains that constitute the secondary residue shells surrounding the active sites also contribute to the kinetic differences (Cavalier et al, 2012). These isoforms show differences in their distribution and kinetic properties in response to allosteric effectors, hormonal, and growth factor signals (Okar et al, 2001).…”

“…This isoform, which has a native activity ratio of roughly 700-fold kinase-to-phosphatase activity, dramatically increases upon phosphorylation of Ser461 by protein kinase A (PKA), AMP-dependent protein kinase (AMPK) or other kinases. The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is solely due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms (Cavalier et al, 2012;Kim et al, 2006). PFKFB3 gene was cloned from a fetal brain library (Manzano et al, 1998;Ventura et al, 1995), human placenta (Sakai et al, 1996) and breast cancer cells (Hamilton et al, 1997).…”