Evolution of Phosphofructokinase.

Comparative sequence data suggest that eucaryotic phosphofructokinase PFK has evolved from a procaryotic precursor by gene duplication, fusion, and mutation of previous catalytic sites into new regulatory ligand binding sites. We are attempting to duplicate these processes by recombinant DNA technology. The E. coli pfk gene is being recloned into a convenient plasmid. Using a synthetic oligonucleotide that matches the mammalian link region, two E. coli pfk genes will be joined. The properties of the expressed duplex enzyme will be analyzed. Utilizing site-directed mutagenesis, conversion of one of the two catalytic sites to a regulatory site will be carried out. Keywords Protein evolution, Protein engineering.