Isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase : structure, catalysis and regulation

Since the discovery of Fru-2.6-P2 more than ten years ago, a great deal has been learned about its role in the control of glycolysis in mammalian tissues and lower organisms, such as yeast. This in turn led to the recognition of the bifunctional enzyme PFK2/FBPase-2 and an understanding of its regulation at the molecular level.
My own interest in the field started with the study of the role of Fru-2.6-P2 concentration in heart, PFK2/FBPase-2 was purified from bovine heart and its properties were compared with those of the rat liver bifunctional enzyme. On the basis of differences in kinetic properties and regulation by protein kinases, it became apparent that rat liver and bovine heart PFK2/FBPase-2 were distinct isozymes. Other PFK2/FBPase-2 isozymes have since been found in skeletal muscle, testis and brain. Over the last years the work has focused on the understanding of the catalytic mechanisms and regulation of PFK-2 and FBPase-2 at the molecular level, using the techniques of chemical modification and site-directed mutagenesis. One of our goals is to solve the three-dimensional structures of the PFK2/FBPase-2 isozymes by X-ray crystallography. This should lead to a detailed understanding of the catalysis and regulation of the bifunctional enzyme. The sequence similarities between the nbf in the PFK-2 domain and other nucleotide binding proteins, which have been crystallized, together with the homology between FBPase-2 and yeast PGM, whose three-dimensional structure is know, will help to fit the X-ray coordinates of PFK2/FBPase-2 into its crystal structure.
Molecular biology has played an important part in our understanding of the molecular enzymology of PFK2/FBPase-2, firstly by providing amino acid sequences of the PFK2/FBPase-2 isozymes from their cDNAs. This in turn has yielded information on the evolution of the bifunctional enzyme. Secondly molecular biology has been used to identify and characterize the genes for PFK2/FBPase-2 and to study the long-term regulation of the PFK2/FBPase-2 isozymes. Thirdly, it is also the tool used in site-directed mutagenesis, which has led to an understanding of catalysis at the PFK2 and FBPase-2 active sites. The study of the physico-chemical properties and regulation of PFK2/FBPase-2 has provided information on its structure. Once the three-dimensional structure of PFK2/FBPase-2 has been solved, it will hopefully give information on the proximity of amino acid side chains ti the substrates in the PFK2 and FBPase-2 active sites. With this information, one can then go back to molecular biology and do site-directed mutagenesis experiments on the active site residues. At the end of the day, organic chemistry will have to be used to provide the molecular mechanisms of the PFK2 and FBPase-2 reactions. The three-dimensional structure of PFK2/FBPase-2 will provide a model of the folding of the PFK-2 and FBPase-2 domains, which, when compared with the topology of proteins, which share sequence similarity with PFK2 and FBPase-2, could yield important information on the evolution of the bifunctional enzyme.
Finally, using the techniques of molecular biology, the PFK2/FBPase-2 isozymes could be expressed in different cell lines to study the role of Fru-2.6-P2 in controlling glycolysis. By expressing, in eukaryotic cells, PFK2/FBPase-2 carrying mutations in the PFK2 or FBPase-2 active sites, one will be able to carry out quantitative studies ti measure the degree of control exerted of whether elevated Fru-2.6-P2 concentrations are necessary for the proliferation of cancer cells. Clearly there is much work still to be done! Biochemistry, the chemistry of life, has taken us from the animal down to the molecule and back to the intact cell. The trip is fascinating, and, fortunately, is still going on