PFK-2/Fbpase-2: Maker and Breaker of the Essential Biofactor Fructose-2, 6-Bisphosphate

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

(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. In the liver, multimodal, effecting the appropriate reciprocal

F-2,6-P2 is derived directly from the fructose- regulation of GK and G-6-Pase, as well as metabolic 6-phosphate/glucose-6-phosphate (F-6-P/G-6-P) pool flux, by reciprocal modulation of PFK-1 and FBPase-1 and is the ultimate messenger in the cyclic AMP activities.

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A B Kinase Bisphosphatase C terminus [F-2,6-P2 ] N terminus K N terminus >1 B Kinase C terminus Bisphosphatase HGP

Protein kinase A Protein phosphatase 2A

Kinase P Bisphosphatase [F-2,6-P ] 2 N terminus C terminus K <1 B N terminus C terminus HGP P Kinase Bisphosphatase

Ti BS

Ti BS Fig. 3. Regulation of the liver bifunctional enzyme activities by post- Fig. 2. A trace of the back-bone structure of the rat testis bifunctional translational modification. Phosphorylation of Ser32 (indicated by the enzyme (2BIF)6. The dimer interface is shown by a solid black line and yellow circle) activates the bisphosphatase and inhibits the kinase. the monomers are designated as A and B. Kinase domains are in red Dephosphorylation of the same residue produces the opposite effects. and green; bisphosphatase domains in blue and magenta. This inversion of the kinase:bisphosphatase (K:B) ratio modulates the hepatic content of fructose-2,6-bisphosphate (F-2,6-P2) as indicated, The liver isoform of the bifunctional enzyme which subsequently effects the hepatic glucose production (HGP). > Glucagon stimulates the cyclic AMP (cAMP)-dependent protein kinase switches between a high K:B state ( 1) and a low K:B A, and glucose stimulates the xylulose-5-phosphate-dependent protein state (<1), depending upon the phosphorylation state phosphatase 2A. Therefore, the opposing metabolic effects of glucose of Ser32 (Fig. 3)17. A decrease in the K:B is due to both and glucagon can be accounted for, at least in part, by their respective a reduction of the kinase activity and an effects on the Ser32 phosphorylation state of 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase. enhancement of the bisphosphatase activity upon phosphorylation of Ser32 in response to glucagon18. transition state analogs provided many insights into

By contrast, upregulation of the F-2,6-P2 levels is the mechanisms of biocatalysis. The bisphosphatase responsive to glucose via stimulation of a xylulose-5- and the serine proteases both use histidine as a phosphate-dependent protein phosphatase that crucial component of their respective catalytic preferentially dephosphorylates Ser32 in the hepatic machinery, however, they employ a different bifunctional enzyme19,20. Although the mechanism constellation of residues at the active site to exploit remains unknown, the effect of Ser32 the amphipathic nature of the imidazole ring to phosphorylation on the K:B appears to be mediated drive different reactions. The 15N-, 13C- and 1H-NMR by the N- and C-termini of the enzyme because spectral signatures of the histidine(s) in these deletion of the N-terminal 22 amino acids and enzyme families allows a comparison of the histidine truncation of the C-terminal 30 amino acids produced at work in two different reaction mechanisms. a bifunctional enzyme that was still phosphorylated, The NMR spectroscopic analyses of the serine but without effect on the activities21. proteases26,27 have revealed strong hydrogen bonds involving the imidazole nitrogens, designated low- Bisphosphatase in detail barrier hydrogen bonds (LBHBs)28. Although the Rarely has it been possible to obtain an X-ray precise role of LBHBs in catalysis remains structure and high-field nuclear magnetic resonance controversial, it is clear that they are significantly (NMR) spectra from an enzyme during turn-over of stronger than the average hydrogen bond. The the physiologically relevant substrate, yet the transition state for hydrolysis of a peptide bond would extreme stability of the covalent phosphohistidine probably be a higher energy state than the stable intermediate of the bisphosphatase has allowed the intermediate for the hydrolysis of an acid-labile sugar acquisition of structural and spectral data22–24. The phosphate. Consistent with this view, the stable intermediate is phosphorylated at the 3′-N of bisphosphatase does not form LBHBs, as judged by His258 and is complexed with F-6-P; the the criteria of the chemical shifts for nuclei within the dissociation of which appears to be the rate-limiting histidine ring during turnover23. Conversely, the 15N, step in the bisphosphatase reaction7,25. As such, the 13C and 1H chemical shifts of His258, phosphorylated bisphosphatase during turnover is similar to the or not, and the adjunct His392, correspond well with serine proteases, where the detailed structural and another phosphohistidine-containing spectral studies of the enzymes in complex with phosphotransferase, IIIGlc of Escherichia coli (Ref. 29).

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Table 1. Bifunctional isozyme propertiesa

PFK-2 FBPase-2 µΜ µΜ Km ( )Km ( )

Isozymes Amino acids Mr Vmax F-6-P ATP Vmax F-2,6-P2 Kinase/ Refs (mU/mg) mU/mg phosphatase Rat liver 470 55 113 35 150 45 <0.1 2.5 12 Bovine liver 470 55 42 150 12 35 7 1.2 45 Fetal rat liver ND 55 80 44 195 ND ND ND 46 Rat muscle 450 54 66 56 48 154 0.4 0.4 47 Bovine heart 570 58 61 74 0.26 33 40 1.8 48 Rat testis 468 55 90 85 270 22 21 4.1 30 Human testis 468 55 75 58 650 80 16 0.9 49 Bovine brain ND 120 90 27 55 29 70 3.1 50 Human placenta 520 59 142 32 220 0.2 130 710 49 a Abbreviations: F-2,6-P2, fructose-2,6-bisphosphate; F-6-P, fructose-6-phosphate; FBPase-2, fructose-2,6-bisphosphatase; ND, none detected or not determined; PFK-2, 6-phosphofructo-2-kinase.

Different needs, different isozymes dimer, the terminal regions can elicit their Since the discovery of rat liver PFK-2/FBPase-2, regulatory function by changes in the tertiary or many more mammalian isozymes have been quaternary conformations in response to the identified in skeletal muscle, heart and brain, as multiplicity of effectors. The localization of the well as a ubiquitous isozyme that is present in dimerization site within the kinase domain and the placenta and tumor cells. The testis30 and the proximity of the Ser32 phosphorylation site favors a liver12 isozymes are the most similar (75% change in quaternary structure as the general identity), followed by liver and heart31 (73% scheme by which the K:B might be inverted. identity). The ubiquitous, or placental, The gene expression of PFK-2/FBP-2 is regulated PFK-2/FBPase-2 shows the greatest divergence by hormones and metabolites12, yet the expression of from the liver isozyme. Although the core structure tissue-specific isoforms of PFK-2/FBPase-2 in their of the different PFK-2/FBPase-2 enzymes is highly respective tissues is not completely exclusive. In conserved, there are differences in kinase and liver, for example, 10% of the expression is from the bisphosphatase properties among the different skeletal muscle isoform32. Also, within tissues that isozymes (Table 1), presumably, due to the are made up of different cell types, different isoforms structural variations in the terminal regions. of the bifunctional enzyme are expressed. The The bifunctional enzyme monomer can be placental/ubiquitous isoform, and not the liver thought of as being composed of four regions, with isoform, is expressed only in the Kupffer cells of the the core kinase and bisphosphatase domains at the liver33. The expression of more than one isozyme in center, and regulatory regions at the termini. These the same tissue suggests that different isozymes play observations are borne out by comparison of the key roles in different physiological conditions or in PFK-2/FBPase-2 amino acid sequences, which show response to specific hormones. high identity in the core catalytic regions. At least six different isoforms of the bifunctional Alignments of the bifunctional enzyme isozymes enzyme have been identified in mammals and all are can be accessed at the Protein Information generated by alternative splicing of the transcribed Resource (PIR; http://pir.georgetown.edu/pirwww/). RNA from only four genes, designated PFKFB1–4 Given that the bifunctional enzyme functions as a (Table 2)34. Consistent with the divergence of the

Table 2.Properties of bifunctional isozyme genesa Localization Gene Size (Kb) Human Rat Isozymes mRNA (Kb) Liver 2.1 PFKFB1 (A) 60 (rat/human) Xq27–q28 Xq22–q31 Muscle 1.9 Fetal 2.2

Heart 4/6.8 PFKFB2 (B) 22 (rat/human) 1q31 13q24–q25 Kidney 4/6.8 Pancreatic islets

Placenta 5.4 PFKFB3 14 (human)b 10p15–p14 Ubiquitous 4.8 Inducible

PFKFB4 40 (human)b 3p22–p21 Testis 2.4 aAbbreviation: PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. bDeduced from GenBank data and Refs 51, 52.

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amino acid sequences in the terminal regions, it is although E. coli do not express any PFK-2 activity,

the use of alternative exons in processing the and therefore do not contain any F-2,6-P2, their primary transcript to generate various mRNAs that PFK-1 is still activated by F-2,6-P2 via the same give rise to the numerous isoforms30,35–39. For allosteric mechanism demonstrated in the example, the rat liver, muscle and fetal isozymes are mammalian enzymes. transcribed from the same gene, but the liver protein Among the eukaryotes, plants, yeast and animals

contains a consensus phosphorylation site for cAMP- appear to have adapted the use of F-2,6-P2 in different 12 dependent protein kinase (Ser32) , whereas the ways. Plants use F-2,6-P2 to partition carbohydrate others do not36. This is consistent with physiological stores between sucrose and starch, which is somewhat demand, because when the liver is exporting glucose analogous to the role of the compound in mammals 16 and the peripheral tissues are using it, the F-2,6-P2 (i.e. to direct fuel use and storage) . Modulation of the level in the liver must be relatively low to minimize bifunctional enzyme K:B is similar in plants, fish, glycolysis, and that in the peripheral tissue must be reptiles, amphibia and mammals5. Yeast PFK-1 is

high to promote it. This balance can be maintained activated by F-2,6-P2 (Ref. 42), however as mentioned only if the relevant bifunctional enzymes are earlier, the yeast have developed a different means to

differentially regulated. control cellular F-2,6-P2 content.

Evolutionary design of a bifunctional enzyme Conclusion and prospects

It appears that the use of F-2,6-P2 as a regulatory We have attempted to show that since its discovery in metabolite is a specifically eukaryotic the early 1980s, the study of PFK-2/FBPase-2 has led phenomenon. The most plausible hypothesis for the to a significant expansion in the number of genes and origin of the PFK-2/FBPase-2 would be the fusion isozymic gene products, as well as an increase in of two ancestral genes coding for a kinase structural and regulatory (both acute and long-term) functional unit and a phosphohydrolase/mutase information about this enzyme. The detailed research 14 unit, respectively . From protein sequence into the F-2,6-P2 system by a variety of methods and alignments, it is clear that the bisphosphatase in several tissues and organisms has illuminated a activity located in the C-terminal domain of the complex set of interwoven control mechanisms that

PFK-2/FBPase-2 is homologous to the make the cellular F-2,6-P2 content responsive to phosphoglycerate mutases (PGMs) and the acid many signals derived from the pathways of fuel 40 phosphatase family . Alignments of the metabolism. Elucidating the role of F-2,6-P2 in the bisphosphatase domain with PGM and acid regulation of hepatic fuel usage in the gilthead sea phosphatase can be accessed at the PIR website bream, a commercial fish important to the economies (http://pir.georgetown.edu/cgi-bin/pirwww/ of the Mediterranean region, is one such example43. nbrfget?uid=DA3761&db=A). The N-terminal Fundamental questions that have yet to be answered PFK-2 domain is sequentially and structurally include: What are the specific roles of the many homologous to several nucleotide binding proteins, isoforms and how are different isozymes regulated primarily that of adenylate kinase of E. coli both acutely and by changes in gene expression? (Refs 6,41; Fig. 4). Furthermore, how do the two domains, kinase and Clearly, the emergence of a discrete nucleus in the bisphosphatase, communicate with each other to early eukaryotes must have required the affect the reciprocal regulation?

development of new metabolic control mechanisms to The importance of F-2,6-P2 to the regulation of augment those of the prokaryotic cells from which it carbohydrate metabolism has made the bifunctional

had so recently evolved. Apparently, F-2,6-P2 was one enzyme a target for therapeutic intervention in biofactor that could fill this role. Interestingly, diabetes. Liver-targeted genetic delivery systems using chimeraplasty44 are being tested in the development of blood glucose-lowering therapies NMPK Phosphohydrolase/ using diabetic mouse models16. In heart, cardiac mutase expression-specific bifunctional enzyme transgenes are under development in an attempt to raise PFK-1 glycolytic rates to prevent diabetes-associated Fig. 4. Convergent and cardiomyopathy(Q. Liang and P.N. Epstein, divergent aspects of PFK-2/FBPase-2 evolution PFK-2 unpublished). These efforts, as well as the in-depth and gene fusion. studies of the structure and mechanism of the Abbreviations: FBPase-2, Mutase bisphosphatase domain, provide insight into the fructose- function of this enzyme at the cellular and molecular 2,6-bisphosphatase; level23,24. It is hoped that the study of the bifunctional NMPK, nucleoside Gene fusion monophosphate kinase; Acid phosphatase enzymes and their genes will benefit those afflicted PFK-1, 6-phosphofructo-1- with diabetes, while we gain a deeper understanding kinase; PFK-2, PFK-2/FBPase-2 of how organisms coordinately regulate the metabolic 6-phosphofructo-2- Ti BS kinase. pathways of life.

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