30 Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001 PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2, 6-bisphosphate DavidA. Okar, Ànna Manzano,Aurèa Navarro-Sabatè, Lluìs Riera, Ramon Bartrons and Alex J. Lange Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated extraction in base led to the discovery of F-2,6-P2 and gluconeogenesis in the liver.This discovery has led to the realization that this the realization that it was either formed or destroyed compound plays a significant role in directing carbohydrate fluxes in all during metabolic transitions where its concentration eukaryotes. Biophysical studies of the enzyme that both synthesizes and determined changes in glycolytic and gluconeogenic degrades this biofactor have yielded insight into its molecular enzymology. carbon flux in the liver. Interest in this compound grew Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential rapidly because it was found to be a most potent in the treatment of diabetes. positive allosteric effector of 6-phosphofructo-1- kinase (PFK-1; EC 2.7.1.11) and an inhibitor of A trail that began with the discovery of a highly potent fructose-1,6-bisphosphatase (FBPase-1; EC 3.1.3.11). regulator of mammalian hepatic carbohydrate Because of its antagonistic actions on these enzymes, metabolism, fructose-2,6-bisphosphate (F-2,6-P2), led F-2,6-P2 plays a crucial role in the control of the to the discovery of the bifunctional enzyme opposing hepatic glycolytic and gluconeogenic 6-phosphofructo-2-kinase/fructose- pathways1–4 (Fig. 1). Because glycolysis requires the 2,6-bisphosphatase (PFK-2/FBPase-2) that is presence of F-2,6-P2, it is important in all glycolysis- responsible for both the formation and degradation of dependent tissues and it has since been found in this compound, and to the genes that code for it. Since virtually every eukaryotic tissue or cell examined5. the discovery of this system in liver, other mammalian Whether it is a fungus switching to an alternative tissue-specific bifunctional isozymes and their genes carbon source, a turtle hatchling freezing in a nest or a have been identified. F-2,6-P2 and the enzymes germinating seed, F-2,6-P2 is intimately involved in the responsible for controlling the amount of this biofactor metabolic fine tuning required for survival. appear to be present in all eukaryotes, including A single enzyme family is responsible for plants and yeast. Although the particular function of determining the levels of F-2,6-P2 by synthesizing and F-2,6-P2 in each cell type varies to some extent, degrading this compound at distinct active sites. That adaptation to changing environmental or metabolic is, the kinase (EC 2.7.1.105) synthesizes F-2,6-P2 from situations is a common theme and this implies that a ATP and fructose-6-phosphate (F-6-P), whereas the diversity of mechanisms have evolved to control the bisphosphatase (EC 3.1.3.46) degrades F-2,6-P2 to relative kinase (synthetic) and bisphosphatase F-6-P and inorganic phosphate (Pi). It should be (degradative) activities. The most striking example of appreciated not only that the enzyme catalyzes this diversity is the yeast, which do not express a reciprocal reactions, but also that it does so as a dimer bifunctional enzyme, rather they use a set of enzymes that is stabilized by numerous protein–protein David A. Okar 6 Alex J. Lange* that have one or the other activity diminished by interactions between the kinase domains (Fig. 2) . By Dept of Biochemistry, ‘mutation’ of key catalytic amino acid residues in the contrast, the crystal structure of the rat testis Molecular Biology and kinase or bisphosphatase active sites. This highlights bifunctional enzyme suggests that the bisphosphatase Biophysics, University of Minnesota, Minneapolis, the complexity of this convoluted signaling enzyme domains have little, if any, contact across the dimeric MN 55455, USA. system, which is an essential determinant in the interface. This view is supported by the observation *e-mail: regulation of carbohydrate metabolism. that the separately expressed rat liver bisphosphatase [email protected] domain is monomeric, even at greater than 4 mM Ànna Manzano A multimodulated bifunctional enzyme (80 mg ml−1)7. The quaternary structure of the rat Aurea Navarro-Sabatè F-2,6-P was discovered during the search for the bifunctional enzyme is relevant to the monofunctional Lluìs Riera 2 Ramon Bartrons mechanism by which glucagon stimulates hepatic yeast isozymes because they retain the two domain 8,9 Unitat de Bioquímica, gluconeogenesis. The fact that it does not participate as structure of the monomers and are dimeric . Campus de Bellvitge, an intermediary in any metabolic interconversion, as However, the ‘monofunctionality’ refers to the subunits Universitat de Barcelona, well as its lability in acid extracts used in systematic of the dimer. Considering that the expression of a C/Feixa Llarga, S/N, 08907 L’Hospitalet, Barcelona, surveys of phosphoric acid esters in tissues, explains single yeast isozyme is not exclusive, it is possible that Spain. why it escaped discovery until 1980. Eventually, the yeast enzymes demonstrate bifunctionality by http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01699-6 Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001 31 (cAMP)-dependent protein kinase cascade set off by Glucose glucagon binding to the extracellular site of its receptor. This compound forms the juncture between the hormonal signal pathway and the metabolic pathway. GK Glu-6-Pase Because both the Km of PFK-2 and the Ki of FBPase-2 for F-6-P are in the physiological range, any alteration of F-6-P concentration should cause inverse changes in the two activities. The reciprocal modulation of the Glucose 6-P kinase and bisphosphatase activities forms the current paradigm for explaining how the bifunctional enzyme can closely regulate the level of F-2,6-P in vivo. Fructose 6-P 2 The kinase reaction proceeds by a sequential- PFK-2/FBPase-2 ordered mechanism for transfer of the phosphate from ATP to F-6-P. The bisphosphatase reaction proceeds via K K PKA KK a covalent phosphohistidine intermediate formed upon Citrate Gly-3-P Gly-3-P_ BB reaction with F-2,6-P . In isolation, neither reaction is BB + Pi 2 PEP freely reversible. The steady state concentration of PP2A F-2,6-P is determined by the balance between these PFK-1 FBPase-1 2 opposing reactions. This kinase:bisphosphatase activity Fructose 2,6-P2 _ ratio (K:B) is the most salient aspect of any given + bifunctional enzyme isoform, whether the active sites reside on the same monomeric component or not, because the balance between the reciprocal reactions is Fructose 1,6-P what determines the net effect on the cellular content of + 2 F-2,6-P2. The K:B is determined by which isoforms are present, the levels of several glycolytic and PEP gluconeogenic metabolites, post-translational PEPCK modification of the enzyme, and even xylulose-5- phosphate, an intermediate of the hexose PK OAA monophosphate pathway. Metabolites beyond PFK-1, such as α-glycerol phosphate, phosphoenolpyruvate and citrate decrease the activity of PFK-2 or favor that of FBPase-2, in accordance with Pyruvate the concept of a negative feed-back control loop11,12. In addition, the bisphosphatase activity is modulated by Lactate, alanine Ti BS GTP and ATP, which activate FBPase-2 at subsaturating substrate concentrations, but inhibit Fig. 1.The position of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) in competitively at saturating substrate concentrations13. the pathways of hepatic intermediary carbohydrate metabolism. Regulation of PFK-2/FBPase-2 The vast range of signals impinging upon the target activities, 6-phosphofructo-1-kinase (PFK-1) and fructose-1,6-bisphosphatase (FBPase-1), as bifunctional enzyme constitute the enzyme’s well as regulation of PFK-2/FBPase-2 itself via effectors and covalent modification are shown. Abbreviations: Gly-3-P, glyceraldehyde-3-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; multimodality and, together, determine the overall K:B PEPCK, PEP carboxykinase; P , inorganic phosphate; PK, pyruvate kinase; PKA, protein kinase A; i and thereby the F-2,6-P2 content of the living PP2A, protein phosphatase 2A. 1–5,11,12,14,15 cell . The significance of F-2,6-P2 with regard forming mixed dimers in which the monomers possess to carbohydrate flux has been exploited to lower the reciprocal kinase and bisphosphatase activities. blood glucose levels in streptozotocin-treated mice, Several years ago, Sols et al. described the concept of which model type I diabetes. This was done using enzyme multimodulation arising from the adenovirus-mediated overexpression of the rat liver accumulation of several regulatory mechanisms in a bifunctional enzyme engineered to have a high K:B given enzyme10. These regulatory mechanisms can be of (Ref. 16). The engineered enzyme has two mutations, different types (cooperative, allosteric or Ser32Ala and His258Ala, which remove a regulatory interconversion), of the same type (multiple allosteric phosphorylation site and a key component of the effects) or of any combination. PFK-2/FBPase-2 has bisphosphatase active site, respectively. many of these properties, making it responsive to a Overexpression of the high K:B bifunctional enzyme plethora of metabolic and hormonal signals. This is markedly stimulated glucokinase (GK) expression and consistent with its central role in regulation of diminished that of glucose-6-phosphatase (G-6-Pase). carbohydrate metabolic fluxes in a diverse cross-section This indicates that the cellular effects of F-2,6-P2 are of tissue types and eukaryotic life-forms.