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Molecular basis of the fructose‐2,6‐bisphosphatase reaction of PFKFB3: Transition state and the C‐terminal function
Author(s) -
Cavalier Michael C.,
Kim SongGun,
Neau David,
Lee YongHwan
Publication year - 2012
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24015
Subject(s) - chemistry , stereochemistry , acceptor , hydrogen bond , ground state , leaving group , crystallography , catalysis , molecule , physics , atomic physics , organic chemistry , condensed matter physics , biochemistry
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•AlF 4 ), and in a complex with pyrophosphate (PFKFB3•PP i ) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3‐P•F‐6‐P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF 4 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 ∼5.1 Å, 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 NP bond, breaking the 2OP 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 oxygen 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 2012; © 2011 Wiley Periodicals, Inc.