Evolution of phosphofructokinase—gene duplication and creation of new effector sites

Abstract

Phosphofructokinases (PFK; EC 2.7.1.11) are tetrameric enzymes that have a key role in the regulation of glycolysis; as such, they are subject to allosteric activation and inhibition by various metabolites. Eukaryotic PFKs are about twice the size of prokaryotic enzymes and are regulated by a wider repertoire of effectors: for example, the subunit molecular weights of rabbit muscle (RM) PFK and Bacillus stearothermophilus (Bs) PFK are 82,000 and 36,000, respectively. Both enzymes are activated by ADP (or AMP), but RM-PFK is also activated by fructose bisphosphates (FBP) and inhibited by ATP and citrate. This, together with other evidence, has led to speculation that mammalian PFKs have evolved by duplication of a prokaryotic gene, although previous peptide analysis failed to reveal internal homology in RM-PFK. Here we demonstrate clear homology among the N- and C-halves of RM-PFK and Bs-PFK, thus establishing an evolutionary relationship by series gene duplication and divergence. Furthermore, detailed knowledge of the Bs-PFK structure provides the basis for inferences concerning the structural organization of RM-PFK and the evolution of new effector sites in the enzyme tetramer.

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

“…On the other hand, substitution of Ser 724 by the charged aspartate completely abolished activation by fructose 2,6-bisphosphate, indicating that the 2-phosphate group may be localized near this residue, confirming its hypothetical role deduced from sequence comparisons (20). Previously, we have shown that mutations abolishing catalytical functions in only one of the genes reduced specific Pfk activity measured in crude extracts by about half (25).…”

“…Activities-Due to a hypothesis on the evolution of eukaryotic Pfk enzymes based on sequence comparisons and deductions from x-ray studies on prokaryotic Pfk, a serine at position 724 and a histidine at position 859 (for numbering and designations of mutant alleles refer to "Materials and Methods") could be important in binding of the allosteric activator fructose 2,6-bisphosphate by yeast Pfk (20,25). To provide evidence for such a binding domain in a eukaryotic Pfk we altered the respective codons by in vitro mutagenesis in both yeast PFK genes (Fig.…”

“…Comparison of the primary sequence of Bacillus stearothermophilus Pfk with the one from rabbit muscle led to the notion that the latter evolved through a gene duplication event from a prokaryotic ancestor (20). This was also postulated for each of the yeast Pfk subunits, where apparently a second gene duplication followed, locating the two genes on different chromosomes (13).…”

“…allosteric) functions could be defined (21,22). It has been speculated that the N-terminal half of rabbit muscle Pfk retained its catalytical function, whereas in the C-terminal half a former fructose 6-phosphate binding site has evolved to a fructose 2,6-bisphosphate binding domain with allosteric function (20). As highly homologous regions have been conserved from bacterial through yeast and human Pfk (23) it seems feasible to deduce functions of specific amino acids in the eukaryotic enzymes.…”

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

“…These results are very similar to those obtained recently by Cronin and Tipton [17] (49 kDa and 222 f 4 kDa, respectively). The molecular mass of trypanosomal phosphofructokinase is apparently unique and, therefore, difficult to fit into the hypothesis put forward by Poorman et al [24], which suggests that mammalian phosphofructokinase (with a subunit mass of 82 kDa) arose by subsequent gene duplication and fusion from an ancestral gene with a coding length equivalent to that of the bacterialtype enzyme (a similar hypothesis for mammalian hexokinases has been cited above).…”