Capillary electrophoresis-based assay of phosphofructokinase-1.

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²âº to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

The crystal structures of eukaryotic phosphofructokinases from baker's yeast and rabbit skeletal muscle.

Phosphofructokinase 1 (PFK) is a multisubunit allosteric enzyme that catalyzes the principal regulatory step in glycolysis-the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by ATP. The activity of eukaryotic PFK is modulated by a number of effectors in response to the cell's needs for energy and building blocks for biosynthesis. The crystal structures of eukaryotic PFKs-from Saccharomyces cerevisiae and rabbit skeletal muscle-demonstrate how successive gene duplications and fusion are reflected in the protein structure and how they allowed the evolution of new functionalities. The basic framework inherited from prokaryotes is conserved, and additional levels of structural and functional complexity have evolved around it. Analysis of protein-ligand complexes has shown how PFK is activated by fructose 2,6-bisphosphate (a powerful PFK effector found only in eukaryotes) and reveals a novel nucleotide binding site. Crystallographic results have been used as the basis for structure-based effector design.

Human H+ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1.

The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1-a4. This study investigated two naturally occurring point mutations in a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H+ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1's catalytic activity. In yeast expression studies, protein levels, H+ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H+ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H+ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H+ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.