PPI-DEPENDENT PHOSPHOFRUCTOKINASE FROM THERMOPROTEUS-TENAX, AN ARCHAEAL DESCENDANT OF AN ANCIENT LINE IN PHOSPHOFRUCTOKINASE EVOLUTION

Flux into the glycolytic pathway of most cells is controlled via allos teric regulation of the irreversible, committing step catalyzed by ATP -dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key e nzyme of glycolysis, In some organisms, the step is catalyzed by PPi-d ependent PFK (PPi-PFK; EC 2.7.1.90), which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction revers ible under physiological conditions. We have determined the enzymic pr operties of PPi-PFK from the anaerobic, hyperthermophilic archaeon The rmoproteus tenax, purified the enzyme to homogeneity, and sequenced th e gene. The similar to 100-kDa PPi-PFK from T. tenax consists of 37-kD a subunits; is not regulated by classical effecters of ATP-PFKs such a s ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenet ic analyses of biochemically characterized PFKs grouped the enzymes in to three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFK s from the genus Propionibacterium, plants, and amitochondriate protis ts; whereas group III consists of PFKs with either cosubstrate specifi city, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly sug gest that the group III PFKs originally bound PPi as a cosubstrate.