Phosphofructokinase crystal

Based on high resolution crystal structures, detailed understanding of the molecular principles of allosteric regulation for several enzyme systems could be gleamed. One of the best studied examples is that of phosphofructokinase from Bac. stearothermophilus (Schirmer and Evans, 1990). 

Philip Evans and his coworkers have determined the crystal structures of phosphofructokinase from two species of bacteria, E. coli and Bacillus stearothermophilus. By crystallizing the enzyme in the presence and absence of the substrate and several allosteric effectors, they obtained detailed views of both the T and R conformations. This work has led to an explanation of why phosphofructokinase appears to be constrained largely to all-or-nothing transitions between these two states, rather than adopting a series of intermediate conformations. 

Figure 9.9 shows the crystal structure of two of the subunits of phosphofructokinase from B. stearothermophilus. In the complete enzyme, the subunits are disposed symmetrically about three mutually perpendicular axes. Each of these axes is a twofold symmetry axis, which means that rotating the entire structure by half of a full circle (180°) around the symmetry axis results in an identical structure. This rotation is shown diagramatically in figure 9.10. Because ADP is a product of the enzymatic reaction as well as an allosteric activator, it binds at both the catalytic and allosteric sites (see figs. 9.9 and 9.10). The catalytic site for fructose-6-phosphate in each subunit is at the interface of the subunit with one of its neighbors, and the allosteric site is at the interface with a different neighbor. 

Computer-generated structure of two of the four subunits of phosphofructokinase from Bacillus stearothermophilus. The enzyme, shown as yellow and light blue tubes, was crystallized in the R conformation in the presence of the substrate fructose-6-phosphate (dark blue) and the allosteric activator ADP (pink). The magnesium ions (white/silver spheres), Mg2+, bound to the ADP molecules are also shown. (Copyright 1994 by the Scripps Research Institute/Molecular Graphics Images by Michael Pique using software by Yng Chen, Michael Connolly, Michael Carson, Alex Shah, and AVS, Inc. Visualization advice by Holly Miller, Wake Forest University Medical Center.)... 

Interface between subunits A and D of phosphofructokinase near the catalytic site in (a) the T and (b) the R structures. Crystals of the enzyme in the R state were obtained in the presence of fructose-6-phosphate and ADP (see fig. 9.9) crystals in the T state were obtained in the presence of a nonphysiological allosteric inhibitor, 2-phosphoglycolate. The wavy green line represents part of the boundary between subunits A and D. The heavy green line indicates the polypeptide backbone. The side chains of Glu 161 and Arg 162 are shown in red. Note the inversion of the positions of these side chains in the two structures. (Source From T. Schirmer and P. R. Evans, Structural basis of the allosteric behaviour of phosphofructokinase, Nature 343 140, 1990.)... 

Parmeggiani A, Luft JH, Love DS, Krebs EG (1966), Crystallization and properties of rabbit skeletal muscle phosphofructokinase, J. Biol. Chem. 241 4625-4637. 

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