PFKFB3 in complex with PPi
[F263_HUMAN] Synthesis and degradation of fructose 2,6-bisphosphate.
Publication Abstract from PubMed
The molecular basis of Fructose-2,6-bisphosphatase (F-2,6-P(2) ase) 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*PP(i) ) at resolutions of 2.45A, 2.2A, and 2.3A, 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*PP(i) complexes were obtained by soaking. The PFKFB3*AlF(4) and PFKFB3*PP(i) 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 approximately 5.1A, 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*PP(i) , implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P(2) ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. Proteins 2011. (c) 2011 Wiley-Liss, Inc.
Molecular basis of the Fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function.,Cavalier MC, Kim SG, Neau D, Lee YH Proteins. 2011 Dec 21. doi: 10.1002/prot.24015. PMID:22275052
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.